An isolated antigen binding protein and uses thereof

Abstract

The present application relates to an isolated antigen binding protein comprising at least one CDR in a VH of an amino acid sequence as set forth in SEQ ID NO: 53; and which comprises at least one CDR in a VL of an amino acid sequence as set forth in SEQ ID NO: 49. The present application also relates to a nucleic acid encoding said isolated antigen binding protein, a vector comprising said isolated nucleic acid, a cell comprising said nucleic acid or said vector, a method of making said isolated antigen binding protein, and uses of said isolated antigen binding protein.

Description

[0001] This application is a divisional application of Chinese patent application filed on August 14, 2020, with application number 2020108200514 and invention title "An isolated antigen-binding protein and its use therein". Technical Field

[0002] This application relates to the field of biomedicine, specifically to an isolated antigen-binding protein and its uses. Background Technology

[0003] Cancer is a serious threat to human health, and liver cancer is a type of malignant tumor with a wide range of effects. Liver cancer caused by hepatitis B virus is characterized by a long incubation period, and once it is discovered, it is often already in an advanced stage. Moreover, it progresses rapidly after onset and has a poor prognosis.

[0004] Glypican 3 (GPC3) is a heparan sulfate proteoglycan found on the cell membrane surface and is present in various tumors, particularly liver cancer. In recent years, with advancements in molecular biology, genomics, and proteomics, a series of molecularly targeted drugs have emerged for the treatment of liver cancer. Molecularly targeted therapy targets certain marker molecules overexpressed by tumor cells, selecting specific inhibitors to effectively intervene and inhibit tumor growth, progression, and metastasis. Compared to the three traditional treatment methods—surgery, radiotherapy, and chemotherapy—molecularly targeted therapy can efficiently and selectively kill tumor cells while reducing damage to normal tissues. However, the current efficacy of molecularly targeted cancer treatment remains unsatisfactory and requires further improvement. Summary of the Invention

[0005] This application provides an isolated antigen-binding protein comprising at least one CDR of the VH amino acid sequence as shown in SEQ ID NO:53; and comprising at least one CDR of the VL amino acid sequence as shown in SEQ ID NO:49.

[0006] In some embodiments, the isolated antigen-binding protein has one or more of the following properties:

[0007] 1) Able to use 6×10 -9 M or lower K D Binds to the GPC3 protein, wherein the K D The value was determined by Octet;

[0008] 2) In FACS assays, it can specifically bind to the GPC3 protein on the surface of HepG2 cells and / or Huh7 cells; and,

[0009] 3) It can inhibit tumor growth and / or tumor cell proliferation.

[0010] In some embodiments, the GPC3 protein comprises human GPC3 protein.

[0011] In some embodiments, the human GPC3 protein comprises an amino acid sequence as shown in SEQ ID NO:74.

[0012] In some embodiments, the tumor includes a GPC3-positive tumor.

[0013] In some implementations, the GPC3-positive tumors include liver cancer.

[0014] In some embodiments, the isolated antigen-binding protein comprises HCDR1 in VH as shown in SEQ ID NO:53.

[0015] In some embodiments, the isolated antigen-binding protein comprises HCDR2 in VH as shown in SEQ ID NO:53.

[0016] In some embodiments, the isolated antigen-binding protein comprises HCDR3 in VH as shown in SEQ ID NO:53.

[0017] In some embodiments, the HCDR1 comprises the amino acid sequence shown in X1YX2MH, where X1 can be D or A, and X2 can be A or E.

[0018] In some embodiments, the HCDR1 comprises the amino acid sequence shown in either SEQ ID NO:4 or 12.

[0019] In some embodiments, the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:20.

[0020] In some embodiments, the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:5, 10, and 13.

[0021] In some embodiments, the HCDR3 comprises X1X2X3X4X5X6X7X8X9X 10 X 11 The amino acid sequence shown can be D or T, X2 can be H or R, X3 can be T or F, X4 can be I or Y, X5 can be G or S, X6 can be V or Y, X7 can be G or A, X8 can be A, Y or H, X9 can be F or blank, X 10 Can be D or blank, X 11 It can be 'I' or blank.

[0022] In some embodiments, the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 6, 11, and 14.

[0023] In some embodiments, the isolated antigen-binding protein comprises LCDR1 in VL as shown in SEQ ID NO:49.

[0024] In some embodiments, the isolated antigen-binding protein comprises LCDR2 in VL as shown in SEQ ID NO:49.

[0025] In some embodiments, the isolated antigen-binding protein comprises LCDR3 in VL as shown in SEQ ID NO:49.

[0026] In some embodiments, the LCDR1 includes X1X2X3X4SX5VX6X7X8X9YX 10 X 11 X 12 X 13 The amino acid sequence shown can be T or R, X2 can be G or S, X3 can be T or S, X4 can be S or Q, X5 can be D or L, X6 can be G or H, X7 can be G or S, X8 can be Y or N, X9 can be N or G, X... 10 It can be V or T, X 11 It can be S or Y, X 12 Can be blank or L, X 13 It can be blank or H.

[0027] In some embodiments, the LCDR1 comprises the amino acid sequence shown in either SEQ ID NO:1 or 7.

[0028] In some embodiments, the LCDR2 comprises the amino acid sequence shown in X1X2SX3RX4S, wherein X1 can be D or K, X2 can be V or G, X3 can be N, Y or Q, and X4 can be P or G.

[0029] In some embodiments, the LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO:2, 8, and 15.

[0030] In some embodiments, the LCDR3 comprises X1X2X3X4X5X6X7X8X9X 10The amino acid sequence shown can be S or G, X2 can be S or Q, X3 can be Y or S, X4 can be A or G, X5 can be S or L, X6 can be G or T, X7 can be S or P, X8 can be T or P, X9 can be L or T, X... 10 It can be a V or a blank.

[0031] In some embodiments, the LCDR3 comprises the amino acid sequence shown in either SEQ ID NO:3 or 9.

[0032] In some embodiments, the isolated antigen-binding protein includes an antibody or an antigen-binding fragment thereof.

[0033] In some embodiments, the antigen-binding fragment includes Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv, and / or dAb.

[0034] In some embodiments, the VL of the isolated antigen-binding protein includes frame regions L-FR1, L-FR2, L-FR3, and L-FR4.

[0035] In some embodiments, the C-terminus of the L-FR1 is directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 contains the amino acid sequence shown in either SEQ ID NO:22 or 30.

[0036] In some embodiments, the L-FR1 comprises the amino acid sequence shown in either SEQ ID NO:22 or 30.

[0037] In some embodiments, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 contains the amino acid sequence shown in either SEQ ID NO:23 or 31.

[0038] In some embodiments, the L-FR2 comprises the amino acid sequence shown in either SEQ ID NO:23 or 31.

[0039] In some embodiments, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 contains the amino acid sequence shown in either SEQ ID NO:24 or 32.

[0040] In some embodiments, the L-FR3 comprises the amino acid sequence shown in either SEQ ID NO:24 or 32.

[0041] In some embodiments, the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3, and the L-FR4 contains the amino acid sequence shown in either SEQ ID NO:25 or 33.

[0042] In some embodiments, the L-FR4 comprises the amino acid sequence shown in either SEQ ID NO:25 or 33.

[0043] In some embodiments, the VL of the isolated antigen-binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:46-48.

[0044] In some embodiments, the isolated antigen-binding protein includes an antibody light chain constant region, and the antibody light chain constant region includes a human Igκ constant region.

[0045] In some embodiments, the constant region of the antibody light chain contains the amino acid sequence shown in SEQ ID NO:54.

[0046] In some embodiments, the isolated antigen-binding protein comprises an antibody light chain LC, and the LC comprises the amino acid sequence shown in any one of SEQ ID NO:56-58.

[0047] In some embodiments, the VH of the isolated antigen-binding protein includes frame regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0048] In some embodiments, the C-terminus of H-FR1 is directly or indirectly connected to the N-terminus of HCDR1, and H-FR1 contains the amino acid sequence shown in either SEQ ID NO:26 or 34.

[0049] In some embodiments, the H-FR1 comprises the amino acid sequence shown in either SEQ ID NO:26 or 34.

[0050] In some embodiments, the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 contains the amino acid sequence shown in either SEQ ID NO:27 or 35.

[0051] In some embodiments, the H-FR2 comprises the amino acid sequence shown in either SEQ ID NO:27 or 35.

[0052] In some embodiments, the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 contains the amino acid sequence shown in either SEQ ID NO:28 or 36.

[0053] In some embodiments, the H-FR3 comprises the amino acid sequence shown in either SEQ ID NO:28 or 36.

[0054] In some embodiments, the N-terminus of the H-FR4 is connected to the C-terminus of the HCDR3, and the H-FR4 contains the amino acid sequence shown in either SEQ ID NO:29 or 37.

[0055] In some embodiments, the H-FR4 comprises the amino acid sequence shown in either SEQ ID NO:29 or 37.

[0056] In some embodiments, the VH of the isolated antigen-binding protein comprises the amino acid sequence shown in any one of SEQ ID NO:50-52.

[0057] In some embodiments, the isolated antigen-binding protein includes an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from the human IgG heavy chain constant region.

[0058] In some embodiments, the isolated antigen-binding protein includes an antibody heavy chain constant region, and the antibody heavy chain constant region is derived from the human IgG1 heavy chain constant region.

[0059] In some embodiments, the antibody heavy chain constant region comprises the amino acid sequence shown in SEQ ID NO:55.

[0060] In some embodiments, the isolated antigen-binding protein comprises an antibody heavy chain HC, and the HC comprises the amino acid sequence shown in any one of SEQ ID NO:59-61.

[0061] This application also provides one or more isolated nucleic acid molecules that encode the isolated antigen-binding protein described in this application.

[0062] This application also provides a vector comprising the nucleic acid molecules described in this application.

[0063] This application also provides a cell comprising the nucleic acid molecules or the vectors described in this application.

[0064] This application also provides a method for preparing the isolated antigen-binding protein described in this application, the method comprising culturing the cells described in this application under conditions that cause the isolated antigen-binding protein to be expressed.

[0065] This application also provides a pharmaceutical composition comprising the isolated antigen-binding protein described in this application, the nucleic acid molecule described in this application, the carrier described in this application, and / or the cell described in this application, and optionally a pharmaceutically acceptable adjuvant.

[0066] This application also provides the use of the isolated antigen-binding protein described in this application, the nucleic acid molecule described in this application, the carrier described in this application, the cell described in this application, and / or the pharmaceutical composition described in this application in the preparation of a medicament for the prevention, relief, and / or treatment of tumors.

[0067] This application also provides the isolated antigen-binding protein described in this application, the nucleic acid molecule described in this application, the carrier described in this application, the cell described in this application, and / or the pharmaceutical composition described in this application, for the prevention, relief, and / or treatment of tumors.

[0068] This application also provides methods for preventing, alleviating, or treating tumors, the methods comprising administering to a subject in need the isolated antigen-binding protein described in this application, the nucleic acid molecule described in this application, the carrier described in this application, the cell described in this application, and / or the pharmaceutical composition described in this application.

[0069] This application also provides a method for detecting GPC3 in a sample, the method comprising applying the isolated antigen-binding protein described in this application.

[0070] Other aspects and advantages of this application will readily be apparent to those skilled in the art from the detailed description below. Only exemplary embodiments of this application are shown and described in the following detailed description. As will be appreciated by those skilled in the art, the content of this application enables them to make modifications to the disclosed specific embodiments without departing from the spirit and scope of the invention to which this application pertains. Accordingly, the descriptions in the accompanying drawings and specification of this application are merely exemplary and not restrictive. Attached Figure Description

[0071] The specific features of the invention involved in this application are shown in the appended claims. The features and advantages of the invention can be better understood by referring to the exemplary embodiments and drawings described in detail below. A brief description of the drawings is as follows:

[0072] Figure 1 The image shows the C-terminal sequences of wild-type human GPC3 protein and its mutants 1-11, where the underlined sites indicate the amino acids corresponding to the mouse GPC3 protein that have been mutated at those sites.

[0073] Figure 2A The results show the epitope analysis results of wild-type human GPC3 protein or mutants 1-5 used to detect L1H2, L2H6 and L1H6 intact antibodies and GC33 control antibody, respectively. Figure 2B The results show the epitope analysis of mutant 6-11 used to detect L1H2, L2H6, and L1H6 intact antibodies, as well as the GC33 control antibody.

[0074] Figure 3 The results of ADCC activity assays for the intact antibodies L1H2, L2H6, and L1H6 are shown.

[0075] Figure 4 This demonstrates the tumor growth inhibition effect of the intact L1H2 antibody. Detailed Implementation

[0076] The following specific embodiments illustrate the implementation of the invention. Those skilled in the art can easily understand other advantages and effects of the invention from the content disclosed in this specification.

[0077] The following further describes this application: In this invention, unless otherwise stated, the scientific and technical terms used herein have the meanings commonly understood by those skilled in the art. Furthermore, the terms and laboratory procedures related to protein and nucleic acid chemistry, molecular biology, cell and tissue culture, microbiology, and immunology used herein are all widely used terms and routine procedures in their respective fields. Meanwhile, to better understand this invention, definitions and explanations of relevant terms are provided below.

[0078] In this application, the term "isolated" generally refers to something obtained artificially from its natural state. If a substance or component is found in nature as an "isolated" substance, it may be due to an alteration of its natural environment, the isolation of the substance from its natural environment, or both. For example, a certain unisolated polynucleotide or polypeptide may naturally exist in the body of a living animal, and a high-purity identical polynucleotide or polypeptide isolated from this natural state is called isolated. The term "isolated" does not exclude the presence of artificial or synthetic substances, nor does it exclude the presence of other impurities that do not affect the activity of the substance.

[0079] In this application, the term "isolated antigen-binding protein" generally refers to a protein with antigen-binding ability obtained artificially from its natural state. This "isolated antigen-binding protein" may include a portion that binds to an antigen and, optionally, allow the antigen-binding portion to employ a scaffold or framework portion that promotes the antigen-binding portion's conformation for binding to the antigen. The antigen-binding protein may comprise, for example, an antibody-derived protein scaffold or an alternative protein scaffold or artificial scaffold having a transplanted CDR or CDR derivative. Such scaffolds include, but are not limited to, antibody-derived scaffolds containing, for example, mutations introduced to stabilize the three-dimensional structure of the antigen-binding protein, and fully synthetic scaffolds containing, for example, biocompatible polymers. See, for example, Korndorfer et al., 2003, Proteins: Structure, Function, and Bioinformatics, 53(1):121-129 (2003); Roque et al., Biotechnol. Prog. 20:639-654 (2004). In addition, peptide antibody mimics ("PAMs") and scaffolds based on antibody mimics utilizing fibronectin components can be used as scaffolds.

[0080] In this application, the term "K" D (Similarly, "K") D "KD" usually refers to the "affinity constant" or "equilibrium dissociation constant," and is used in titration measurements at equilibrium, or by expressing the dissociation rate constant (k... d Divide by the binding rate constant (k) a The value obtained using the binding rate constant (k) a ), dissociation rate constant (k d ) and equilibrium dissociation constant (K D The term K represents the binding affinity of a binding protein (e.g., the isolated antigen-binding protein described in this application) to an antigen (e.g., the GPC3 protein). Methods for determining binding and dissociation rate constants are well known in the art. The use of fluorescence-based techniques provides high sensitivity and the ability to examine samples at equilibrium in physiological buffers. For example, the K can be determined by Octet. D The value can also be determined using other experimental methods and instruments, such as BIAcore (Biomolecular Interaction Analysis) (e.g., instruments available from BIAcore International AB, aGE Healthcare Company, Uppsala, Sweden). Alternatively, the K value can be determined using KinExA (Kinetic Exclusion Assay) available from Sapidyne Instruments (Boise, Idaho). D value.

[0081] In this application, the term "GPC3 protein" generally refers to glypican 3 (GPC3), a heparan sulfate proteoglycan on the cell membrane surface, present in various tumors, particularly liver cancer. GPC3 protein is highly expressed in the liver during fetal development. Abnormal expression of GPC3 protein after birth is closely related to tumor development and progression. GPC3 protein is highly expressed in primary liver cancer (PHC), while it is expressed at low or moderate levels in other tumors or benign liver diseases. For example, the GPC3 protein described in this application may include human GPC3 protein.

[0082] In this application, the terms "specific binding" or "specific" generally refer to measurable and reproducible interactions, such as binding between a target and an antibody, where the presence of the target can be determined in the presence of a heterogeneous population of molecules (including biomolecules). For example, an antibody that specifically binds to a target (which may be an epitope) binds to that target with greater affinity, strength, ease, and / or duration than it binds to other targets. In one embodiment, the degree to which the antibody binds to an irrelevant target is less than about 10% of the antibody's binding to the target, as measured, for example, by radioimmunoassay (RIA). For example, in this application, the isolated antigen-binding protein is capable of <6 x 10 -9 An M or lower dissociation constant (KD) binds to the GPC3 protein. In some embodiments, the antibody specifically binds to an epitope on the protein, which is conserved across proteins of different species. In another embodiment, specific binding may include, but is not required to be, exclusive binding.

[0083] In this application, the term "inhibition" generally refers to a reduction in the growth rate or number of cells. For example, the isolated antigen-binding protein described in this application can inhibit tumor growth and / or tumor cell proliferation.

[0084] In this application, the term "tumor" generally refers to a growth or solid lesion formed by abnormal cell growth. In this application, a tumor can be a solid tumor or a hematologic malignancy. For example, in this application, a tumor can be a GPC3-positive tumor, wherein a GPC3-positive tumor can include liver cancer.

[0085] In this application, the term "variable domain" generally refers to the amino-terminal domain of the antibody heavy or light chain. The variable domains of the heavy and light chains may be referred to as "VH" and "VL," respectively. These domains are typically the most variable parts of the antibody (relative to other antibodies of the same type) and contain antigen-binding sites.

[0086] In this application, the term "variable" generally refers to the fact that certain segments of the variable domain differ significantly in sequence between antibodies. The V domain mediates antigen binding and determines the specificity of a particular antibody for its specific antigen. However, variability is not uniformly distributed across the entire variable domain. Instead, it is concentrated in three segments within the variable domain of the light and heavy chains, called hypervariable regions (CDRs or HVRs). The more conserved portions of the variable domain are called framework regions (FRs). The variable domains of the natural heavy and light chains each contain four FR regions, most of which adopt a β-sheet configuration, linked by three CDRs that form a ring link and, in some cases, form part of a β-sheet structure. The CDRs in each chain are held together closely by the FR regions, and CDRs from the other chain together promote the formation of the antigen-binding site of the antibody (see Kabat et al, Sequences of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). Constant domains do not directly participate in antibody-antigen binding, but exhibit various effector functions, such as antibody involvement in antibody-dependent cytotoxicity.

[0087] In this application, the term "antibody" generally refers to an immunoglobulin or a fragment thereof or a derivative thereof, encompassing any polypeptide that includes an antigen-binding site, whether it is produced in vitro or in vivo. This term includes, but is not limited to, polyclonal, monoclonal, single-specific, multi-specific, non-specific, humanized, single-chain, chimeric, synthetic, recombinant, hybrid, mutated, and transplanted antibodies. Unless otherwise modified by the term "complete," such as in "complete antibody," for the purposes of this invention, the term "antibody" also includes antibody fragments such as Fab, F(ab')2, Fv, scFv, Fd, dAb, and other antibody fragments that maintain antigen-binding function (e.g., specific binding to GPC3). Typically, such fragments should include an antigen-binding domain. The basic 4-chain antibody unit is a heterotetrameric glycoprotein composed of two identical light (L) chains and two identical heavy (H) chains. IgM antibodies consist of five basic heterotetrameric units and a polypeptide chain called the J chain, containing 10 antigen-binding sites. IgA antibodies consist of 2-5 basic tetrameric units that can polymerize with the J chain to form multivalent combinations. For IgG, a tetrameric unit is typically about 150,000 Daltons. Each L chain is linked to an H chain by a covalent disulfide bond, and two H chains are linked to each other by one or more disulfide bonds depending on the H chain isoform. Each H and L chain also has regularly spaced intrachain disulfide bridging bonds. Each H chain has a variable domain (VH) at its N-terminus, followed by three constant domains (CH) for α and γ chains, and four CH domains for μ and ε isoforms. Each L chain has a variable domain (VL) at its N-terminus and a constant domain at its other end. VL corresponds to VH, and CL corresponds to the first constant domain (CH1) of the heavy chain. Specific amino acid residues are thought to form interfaces between the variable domains of the light and heavy chains. VH and VL pair together to form a single antigen-binding site. For the structure and properties of different classes of antibodies, see, for example, Basic and Clinical Immunology, 8th Edition, Daniel P. Sties, Abba I. Terr and Tristram G. Parsolw (eds), Appleton & Lange, Norwalk, Conn., 1994, p. 71 and Chapter 6. The L chain from any vertebrate species can be classified into one of two distinct types, called κ and λ, based on the amino acid sequence of its constant domain. Immunoglobulins can be classified into different classes or isotypes depending on the amino acid sequence of their heavy chain (CH) constant domain. There are five classes of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, with heavy chains named α, δ, ε, γ, and μ, respectively.Based on relatively small differences in CH sequence and function, γ and α classes are further divided into subclasses. For example, humans express the following subclasses: IgG1, IgG2A, IgG2B, IgG3, IgG4, IgA1, and IgK1.

[0088] In this application, the term "CDR" generally refers to a region of the antibody's variable structural domain, whose sequence is highly variable and / or forms a structurally defined loop. Typically, antibodies comprise six CDRs; three in the VH (HCDR1, HCDR2, HCDR3) and three in the VL (LCDR1, LCDR2, LCDR3). In natural antibodies, HCDR3 and LCDR3 exhibit the majority of the diversity of the six CDRs, and HCDR3 is particularly considered to play a unique role in conferring fine specificity to the antibody. See, for example, Xu et al., Immunity 13:37-45 (2000); Johnson and Wu, in Methods in Molecular Biology 248:1-25 (Lo, ed., Human Press, Totowa, NJ, 2003). In fact, naturally occurring camel antibodies, composed only of heavy chains, function normally and stably in the absence of light chains. See, for example, Hamers-Casterman et al., Nature 363:446-448 (1993); Sheriff et al., Nature Struct.Biol.3:733-736 (1996).

[0089] In this application, the term "FR" generally refers to a more conserved portion of the antibody variable domain, referred to as the frame region. Typically, the variable domains of the natural heavy and light chains each contain four FR regions: four in the VH (H-FR1, H-FR2, H-FR3, and H-FR4) and four in the VL (L-FR1, L-FR2, L-FR3, and L-FR4). For example, the VL of the isolated antigen-binding protein described in this application may include frame regions L-FR1, L-FR2, L-FR3, and L-FR4. The VH of the isolated antigen-binding protein described in this application may include frame regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0090] In this application, the term "antigen-binding fragment" generally refers to a fragment with antigen-binding activity. In this application, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragments, F(ab')2, scFv, di-scFv, and / or dAb.

[0091] In this application, the term "competitive binding" generally refers to the ability of an antibody or fragment thereof to interfere with the ability of another antibody (e.g., a reference antibody) to bind a target / antigen (e.g., GPC3) directly or indirectly by allosterically modulating the other antibody (e.g., a reference antibody). For example, in this application, the isolated antigen-binding protein may compete with the reference antibody for binding to the GPC3 protein. Furthermore, the extent to which an antibody or fragment thereof can interfere with the binding of another antibody or fragment thereof to a target, and therefore whether it can be considered as blocking or competing according to the invention, can be determined using a competitive binding assay. A particularly suitable quantitative competitive assay uses FACS-based or AlphaScreen-based methods to measure the competition between a labeled (e.g., His-labeled, biotinylated, or radiolabeled) antibody or fragment thereof and another antibody or fragment thereof in terms of target binding. Typically, the competing antibody or fragment thereof is, for example, one that binds to the target in a competitive assay such that, during the assay and in the presence of a second antibody or fragment thereof, the recorded substitution of the isolated antigen-binding protein of the present invention reaches at most 100% of the maximum theoretical substitution (e.g., substitution by a cold (e.g., unlabeled) antibody or fragment thereof that needs to be blocked) obtained by a detected potential blocking antibody or fragment thereof present in a given amount (e.g., in a FACS-based competitive assay). Preferably, the competing antibody or fragment thereof has a recorded substitution between 10% and 100%, such as between 50% and 100%.

[0092] In this application, the term "directly linked" is used in contrast to the term "indirectly linked." "Directly linked" generally refers to a direct connection between substances. For example, direct linking can mean that substances are directly linked without a spacer. The spacer can be a linker. For example, the linker can be a peptide linker. The term "indirectly linked" generally refers to a connection between substances that are not directly linked. For example, indirect linking can mean that substances are linked through a spacer. For example, in the isolated antigen-binding protein described in this application, the C-terminus of L-FR1 and the N-terminus of LCDR1 can be directly or indirectly linked.

[0093] In this application, the term "isolated nucleic acid molecule" generally refers to an isolated form of nucleotide, deoxyribonucleotide or ribonucleotide of any length, or an analogue isolated from its natural environment or synthesized artificially.

[0094] In this application, the term "vector" generally refers to a nucleic acid delivery vehicle into which a polynucleotide encoding a protein is inserted, thereby enabling the protein to be expressed. Vectors can be used to transform, transduce, or transfect host cells, allowing the genetic material elements they carry to be expressed within the host cells. Examples of vectors include: plasmids; phage particles; Cos plasmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses. Animal viruses used as vectors include retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). A vector may contain multiple elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, vectors may contain a replication initiation site. The carrier may also include components that help it enter the cell, such as viral particles, liposomes, or protein coats, but not only these substances.

[0095] In this application, the term "cell" generally refers to a single cell, cell line, or cell culture that may be or is already a recipient of a subject plasmid or vector, including the nucleic acid molecules or vectors described in this invention. Cells may include the progeny of a single cell. Due to natural, accidental, or intentional mutations, the progeny may not necessarily be identical to the original parent cell (in terms of the morphology of the total DNA complement or in the genome). Cells may include cells transfected in vitro with the vectors described in this invention. Cells may be bacterial cells (e.g., *E. coli*), yeast cells, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells, or myeloma cells. In some embodiments, the cells are mammalian cells. In some embodiments, the mammalian cells are HEK293 cells.

[0096] In this application, the term "pharmaceutical composition" generally refers to a composition suitable for administration to a patient, preferably a human patient. For example, a pharmaceutical composition described in this application may comprise the isolated antigen-binding protein described in this application, the nucleic acid molecule described in this application, the carrier described in this application, and / or the cell described in this application, and optionally a pharmaceutically acceptable adjuvant. Furthermore, the pharmaceutical composition may also comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0097] In this application, the term "pharmaceuticalally acceptable adjuvant" generally refers to any and all solvents, dispersion media, coatings, isotonic agents, and absorption delay agents that are compatible with drug administration, are generally safe and non-toxic, and are neither biologically nor otherwise undesirable.

[0098] In this application, the term "subject" generally refers to a human or non-human animal, including but not limited to cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

[0099] In this application, the term "comprising" generally means including the explicitly specified features, but does not exclude other elements.

[0100] In this application, the term "about" generally refers to a variation within a range of 0.5% to 10% above or below a specified value, such as a variation within a range of 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5%, or 10% above or below a specified value.

[0101] Isolated antigen-binding proteins

[0102] On the one hand, this application provides an isolated antigen-binding protein containing at least one CDR of the VH amino acid sequence as shown in SEQ ID NO:53; and containing at least one CDR of the VL amino acid sequence as shown in SEQ ID NO:49.

[0103] X1VQLVQSGX2X3X4X5X6PGX7SX8X9X 10 SCX 11 ASGX 12 X 13 FX 14 X 15 YX 16 MHWVRQAPGX 17 GLEWX 18 X19X 20 LX 21 X 22 X 23 X 24 GX 25 X 26 X 27 YX 28 X 29 X 30 X 31 X 32 GRX 33 TX 34 X 35 X 36 DX37 X 38 X 39 X 40 X 41 X 42 YX 43 X 44 X 45 X 46 X 47 LRX 48 X 49 DTX 50 X 51 YYCX 52 X 53 X 54 X 55 X 56 X 57 X 58 X 59 X 60 X 61 X 62 X 63 X 64 WGQGTX 65 VTVSS (SEQ ID NO:53), where X1 can be E or Q, X2 can be G or A, X3 can be G or E, X4 can be L or V, X5 can be V or K, X6 can be Q or K, X7 can be R or A, X8 can be L or V, X9 can be R or K, X 10 It can be L or V, X 11 It can be A or K, X 12 It can be F or Y, X 13 It can be T or D, X 14 It can be D or T, X 15 It can be D or A, X 16 It can be A or E, X 17 It can be K or Q, X 18 It can be V or M, X 19 It can be S or G, X 20 It can be G or A, X 21 It can be S or D, X 22 It can be W or P, X 23 It can be N or K, X 24 It can be S or T, X 25 It can be S or Q, X 26 It can be I or T, X 27 It can be G or A, X 28 It can be A or S, X 29 It can be D or Q, X 30 It can be S or K, X 31 It can be V or F, X 32It can be K or Q, X 33 It can be F or V, X 34 It can be I or L, X 35 It can be S or T, X 36 It can be R or A, X 37 It can be N or K, X 38 It can be A or S, X 39 It can be K or I, X 40 It can be N or S, X 41 It can be S or T, X 42 It can be L or A, X 43 It can be L or M, X 44 It can be Q or E, X 45 It can be M or L, X 46 It can be N or S, X 47 It can be S or R, X 48 It can be A or S, X 49 It can be E or D, X 50 It can be A or V, X 51 It can be L or V, X 52 It can be A or blank, X 53 Can be K or blank, X 54 It can be D or T, X 55 It can be H or R, X 56 It can be T or F, X 57 It can be I or Y, X 58 It can be G or S, X 59 It can be V or Y, X 60 It can be G or A, X 61 It can be A, Y, or H, X 62 It can be F or blank, X 63 Can be D or blank, X 64 It can be I or blank, X 65 It can be M or L.

[0104] For example, the VH of the isolated antigen-binding protein described in this application may contain the amino acid sequence shown in any one of SEQ ID NO:50-52.

[0105] X1X2VX3TQX4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X 19 X 20 X 21X 22 X 23 X 24 SX 25 VX 26 X 27 X 28 X 29 YX 30 X 31 X 32 X 33 WYQQX 34 PGX 35 X 36 PX 37 LX 38 IYX 39 X 40 SX 41 RX 42 SGVX 43 X 44 RFSGSX 45 SGX 46 X 47 X 48 X 49 LX 50 ISX51X 52 X 53 AEDX 54 X 55 X 56 YYCX 57 X 58 X 59 X 60 X 61 X 62 X 63 X 64 X 65 X 66 FGX 67 GTKLX 68 X 69 X 70 (SEQ ID NO:49), where X1 can be Q or D, X2 can be S or V, X3 can be L or M, X4 can be P or S, X5 can be A or P, X6 can be S or L, X7 can be V or S, X8 can be S or L, X9 can be G or P, X 10 It can be S or V, X 11 It can be P or T, X 12 It can be G or L, X 13 It can be Q or G, X 14 It can be S or Q, X 15 It can be I or P, X 16 It can be T or A, X 17 It can be I or S, X 18It can be S or I, X 19 It can be C or S, X 20 Can be blank or C, X 21 It can be T or R, X 22 It can be G or S, X 23 It can be T or S, X 24 It can be S or Q, X 25 It can be D or L, X 26 It can be G or H, X 27 It can be G or S, X 28 It can be Y or N, X 29 It can be N or G, X 30 It can be V or T, X 31 It can be S or Y, X 32 Can be blank or L, X 33 Can be blank or H, X 34 It can be H or R, X 35 It can be K or Q, X 36 It can be A or S, X 37 It can be K or R, X 38 It can be M or L, X 39 It can be D or K, X 40 It can be V or G, X 41 It can be N, Y, or Q, X 42 It can be P or G, X 43 It can be S or P, X 44 It can be N or D, X 45 It can be K or G, X 46 It can be N or T, X 47 It can be T or D, X 48 It can be A or F, X 49 It can be S or T, X 50 It can be T or K, X 51 It can be G or R, X 52 It can be L or V, X 53 It can be Q or E, X 54 It can be E or V, X 55 It can be A or G, X 56 It can be D or V, X 57 It can be S or G, X 58 It can be S or Q, X 59 It can be Y or S, X 60 It can be A or G, X 61 It can be S or L, X 62 It can be G or T, X 63 It can be S or P, X 64 It can be T or P, X 65 It can be L or T, X66 It can be V or blank, X 67 It can be G or S, X 68 It can be T or E, X 69 It can be V or I, X 70 It can be L or K.

[0106] For example, the VL of the isolated antigen-binding protein described in this application may contain the amino acid sequence shown in any one of SEQ ID NO:46-48.

[0107] For example, in this application, the isolated antigen-binding protein may contain HCDR1 in the VH amino acid sequence as shown in SEQ ID NO:53. For example, in this application, the isolated antigen-binding protein may contain HCDR2 in the VH amino acid sequence as shown in SEQ ID NO:53. For example, in this application, the isolated antigen-binding protein may contain HCDR3 in the VH amino acid sequence as shown in SEQ ID NO:53. As another example, in this application, the isolated antigen-binding protein may contain LCDR1 in the VL amino acid sequence as shown in SEQ ID NO:49. For example, in this application, the isolated antigen-binding protein may contain LCDR2 in the VL amino acid sequence as shown in SEQ ID NO:49. For example, in this application, the isolated antigen-binding protein may contain LCDR3 in the VL amino acid sequence as shown in SEQ ID NO:49.

[0108] The properties of the isolated antigen-binding protein

[0109] In this application, the isolated antigen-binding protein may have one or more of the following properties:

[0110] 1) Able to use 6×10 -9 M or lower K D Binds to the GPC3 protein, wherein the K D The value was determined by Octet;

[0111] 2) In FACS assays, it can specifically bind to the GPC3 protein on the surface of HepG2 cells and / or Huh7 cells; and,

[0112] 3) It can inhibit tumor growth and / or tumor cell proliferation.

[0113] In this application, the isolated antigen-binding protein is capable of being separated at 6 × 10⁻⁶. -9 M or lower K D Binds to the GPC3 protein, wherein the K DThe value can be determined using Octet. For example, the isolated antigen-binding protein described in this application binds to K-derived human GPC3 protein. D The value can be ≤6x10 -9 M, ≤5x10 -9 M, ≤4x10 -9 M, ≤3x10 -9 M, ≤2x10 -9 M, ≤1x10 -9 M, ≤1x10 -10 M, ≤2x10 -10 M, ≤3x10 -10 M, ≤4x10 -10 M, ≤5x10 -10 M, ≤6x10 -10 M, ≤7x10 -10 M, ≤8x10 -10 M, ≤9x10 -10 M. For example, the isolated antigen-binding protein described in this application binds to the K-type GPC3 protein derived from mice. D The value can be ≤6x10 -9 M, ≤5x10 -9 M, ≤4x10 -9 M, ≤3x10 -9 M, ≤2x10 -9 M, ≤1x10 -9 M, ≤1x10 - 10 M, ≤2x10 -10 M, ≤3x10 -10 M, ≤4x10 -10 M, ≤5x10 -10 M, ≤6x10 -10 M, ≤7x10 -10 M, ≤8x10 -10 M, ≤9x10 -10 M. For example, the isolated antigen-binding protein described in this application binds to the K-type GPC3 protein derived from monkeys. D The value can be ≤6x10 -9 M, ≤5x10 -9 M, ≤4x10 -9 M, ≤3x10 -9 M, ≤2x10 -9 M, ≤1x10 -9 M, ≤1x10 -10 M, ≤2x10 -10 M, ≤3x10 -10 M, ≤4x10 -10 M, ≤5x10 -10 M, ≤6x10-10 M, ≤7x10 -10 M, ≤8x10 -10 M, ≤9x10 -10 M.

[0114] In this application, the K D The value can also be determined by ELISA, competitive ELISA, BIACORE, or KINEXA.

[0115] In this application, the isolated antigen-binding protein is capable of specifically binding to GPC3 protein on the surface of HepG2 cells and / or Huh7 cells, and this specific binding can be measured by FACS. For example, the half-maximal effective concentration (EC50) in the FACS measurement can reflect the specific binding of the isolated antigen-binding protein to GPC3 protein on the surface of HepG2 cells and / or Huh7 cells; for example, a lower EC50 indicates better specific binding. For example, the EC50 value of the isolated antigen-binding protein binding to GPC3 protein on the surface of HepG2 cells and / or Huh7 cells in FACS assay can be 0.01 μg / ml to 0.20 μg / ml, 0.01 μg / ml to 0.02 μg / ml, 0.01 μg / ml to 0.03 μg / ml, 0.01 μg / ml to 0.04 μg / ml, 0.01 μg / ml to 0.05 μg / ml, 0.01 μg / ml to 0.06 μg / ml, 0.01 μg / ml to 0.07 μg / ml, 0.01 μg / ml to 0.08 ... .09μg / ml, 0.01μg / ml~0.10μg / ml, 0.01μg / ml~0.11μg / ml, 0.01μg / ml~0.12μg / ml, 0.01μg / ml~0.13μg / ml, 0.01μg / ml~0.14μg / ml, 0.01μ g / ml~0.15μg / ml, 0.01μg / ml~0.16μg / ml, 0.01μg / ml~0.17μg / ml, 0.01μg / ml~0.18μg / ml, 0.01μg / ml~0.19μg / ml or 0.01μg / ml~0.20μg / ml.

[0116] In this application, the isolated antigen-binding protein described herein is capable of inhibiting tumor growth and / or tumor cell proliferation, for example, reducing tumor volume by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99%, or 100%. Similarly, it is capable of reducing the number of tumor cells by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 99%, or 100%.

[0117] In this application, the GPC3 protein may comprise human GPC3 protein, which may comprise an amino acid sequence as shown in SEQ ID NO:74.

[0118] In this application, the tumor may include a GPC3-positive tumor, and the GPC3-positive tumor may include liver cancer.

[0119] In this application, the isolated antigen-binding protein can inhibit tumor cell growth by inducing antibody-dependent cell-mediated cytotoxicity (ADCC). For example, the isolated antigen-binding protein can carry cytotoxic infiltrating T lymphocytes into tumor tissue, thereby inhibiting tumor cell growth. As another example, the isolated antigen-binding protein can also indirectly inhibit the proliferation of GPC3-positive cells through macrophage-related mechanisms.

[0120] In this application, the isolated antigen-binding protein possesses a certain degree of endocytic activity. This endocytic activity can be detected using the Confocal method.

[0121] The types of isolated antigen-binding proteins

[0122] In this application, the isolated antigen-binding protein may include an antibody or an antigen-binding fragment thereof. For example, the isolated antigen-binding protein described in this application may include, but is not limited to, recombinant antibodies, monoclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, bispecific antibodies, single-chain antibodies, biantibodies, triantibodies, tetraantibodies, Fv fragments, scFv fragments, Fab fragments, Fab' fragments, F(ab')2 fragments, and camelified single-domain antibodies.

[0123] In this application, the antibody may be a humanized antibody. In other words, the isolated antigen-binding protein described in this application may be an antibody or its variants, derivatives, analogs, or fragments thereof that specifically binds to the relevant antigen (e.g., human GPC3) and comprises a frame (FR) region having substantially the amino acid sequence of a human antibody and a complementarity-determining region (CDR) having substantially the amino acid sequence of a non-human antibody. Here, "substantially" in the case of a CDR means that the amino acid sequence of the CDR is at least 80%, preferably at least 85%, at least 90%, at least 95%, at least 98%, or at least 99% identical to the amino acid sequence of the non-human antibody CDR. The humanized antibody may substantially comprise all at least one and typically two variable domains (Fab, Fab′, F(ab′)2, FabC, Fv), wherein all or substantially all CDR regions correspond to the CDR regions of non-human immunoglobulins (i.e., antibodies) and all or substantially all frame regions are frame regions having a common sequence of human immunoglobulins. Preferably, the humanized antibody further comprises at least a portion of an immunoglobulin constant region (e.g., Fc), typically a constant region of human immunoglobulins. In some embodiments, the humanized antibody contains a light chain and at least a variable domain of the heavy chain. The antibody may also include CH1, hinge, CH2, CH3, and CH4 regions of the heavy chain. In some embodiments, the humanized antibody contains only the humanized light chain. In some embodiments, the humanized antibody contains only the humanized heavy chain. In certain embodiments, the humanized antibody contains only the humanized variable domain of the light chain and / or the humanized heavy chain.

[0124] In this application, the antigen-binding fragment may include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and / or dAb.

[0125] CDR

[0126] In this application, the LCDR1 may include X1X2X3X4SX5VX6X7X8X9YX 10 X 11 X 12 X 13 The amino acid sequence shown can be T or R, X2 can be G or S, X3 can be T or S, X4 can be S or Q, X5 can be D or L, X6 can be G or H, X7 can be G or S, X8 can be Y or N, X9 can be N or G, X... 10 It can be V or T, X 11 It can be S or Y, X 12 Can be blank or L, X 13 It can be blank or H.

[0127] In this application, the LCDR1 may contain the amino acid sequence shown in either SEQ ID NO:1 or 7.

[0128] In this application, the LCDR2 may contain the amino acid sequence shown in X1X2SX3RX4S, where X1 can be D or K, X2 can be V or G, X3 can be N, Y or Q, and X4 can be P or G.

[0129] In this application, the LCDR2 may contain the amino acid sequence shown in any one of SEQ ID NO:2, 8 and 15.

[0130] In this application, the LCDR3 may include X1X2X3X4X5X6X7X8X9X 10 The amino acid sequence shown can be S or G, X2 can be S or Q, X3 can be Y or S, X4 can be A or G, X5 can be S or L, X6 can be G or T, X7 can be S or P, X8 can be T or P, X9 can be L or T, X... 10 It can be a V or a blank.

[0131] In this application, the LCDR3 may contain the amino acid sequence shown in either SEQ ID NO:3 or 9.

[0132] For example, the isolated antigen-binding protein LCDR1 described in this application may contain the amino acid sequence shown in SEQ ID NO:1, LCDR2 may contain the amino acid sequence shown in SEQ ID NO:2, and LCDR3 may contain the amino acid sequence shown in SEQ ID NO:3.

[0133] For example, the isolated antigen-binding protein LCDR1 described in this application may contain the amino acid sequence shown in SEQ ID NO:7, LCDR2 may contain the amino acid sequence shown in SEQ ID NO:8, and LCDR3 may contain the amino acid sequence shown in SEQ ID NO:9.

[0134] For example, the isolated antigen-binding protein LCDR1 described in this application may contain the amino acid sequence shown in SEQ ID NO:7, LCDR2 may contain the amino acid sequence shown in SEQ ID NO:15, and LCDR3 may contain the amino acid sequence shown in SEQ ID NO:9.

[0135] In this application, HCDR1 may contain the amino acid sequence shown in X1YX2MH, where X1 can be D or A, and X2 can be A or E.

[0136] In this application, the HCDR1 may contain the amino acid sequence shown in either SEQ ID NO:4 or 12.

[0137] In this application, the HCDR2 may contain the amino acid sequence shown in SEQ ID NO:20.

[0138] X1LX2X3X4X5GX6X7X8YX9X 10 X 11 X 12 X 13 G (SEQ ID NO:20), where X1 can be G or A, X2 can be S or D, X3 can be W or P, X4 can be N or K, X5 can be S or T, X6 can be S or Q, X7 can be I or T, X8 can be G or A, X9 can be A or S, X 10 It can be D or Q, X 11 It can be S or K, X 12 It can be V or F, X 13 It can be K or Q.

[0139] In this application, the HCDR2 may contain the amino acid sequence shown in any one of SEQ ID NO:5, 10 and 13.

[0140] In this application, the HCDR3 may include X1X2X3X4X5X6X7X8X9X 10 X 11 The amino acid sequence shown can be D or T, X2 can be H or R, X3 can be T or F, X4 can be I or Y, X5 can be G or S, X6 can be V or Y, X7 can be G or A, X8 can be A, Y or H, X9 can be F or blank, X 10 Can be D or blank, X 11 It can be 'I' or blank.

[0141] In this application, the HCDR3 may contain the amino acid sequence shown in any one of SEQ ID NO:6, 11 and 14.

[0142] For example, the isolated antigen-binding protein HCDR1 described in this application may contain the amino acid sequence shown in SEQ ID NO:4, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:5, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:6.

[0143] For example, the isolated antigen-binding protein HCDR1 described in this application may contain the amino acid sequence shown in SEQ ID NO:12, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:10, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:11.

[0144] For example, the isolated antigen-binding protein HCDR1 described in this application may contain the amino acid sequence shown in SEQ ID NO:12, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:13, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:14.

[0145] For example, the isolated antigen-binding protein LCDR1 described in this application may contain the amino acid sequence shown in SEQ ID NO:1, LCDR2 may contain the amino acid sequence shown in SEQ ID NO:2, LCDR3 may contain the amino acid sequence shown in SEQ ID NO:3, and HCDR1 may contain the amino acid sequence shown in SEQ ID NO:4, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:5, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:6.

[0146] For example, the isolated antigen-binding protein LCDR1 described in this application may contain the amino acid sequence shown in SEQ ID NO:7, LCDR2 may contain the amino acid sequence shown in SEQ ID NO:8, LCDR3 may contain the amino acid sequence shown in SEQ ID NO:9, and HCDR1 may contain the amino acid sequence shown in SEQ ID NO:12, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:10, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:11.

[0147] For example, the isolated antigen-binding protein LCDR1 described in this application may contain the amino acid sequence shown in SEQ ID NO:7, LCDR2 may contain the amino acid sequence shown in SEQ ID NO:8, LCDR3 may contain the amino acid sequence shown in SEQ ID NO:9, and HCDR1 may contain the amino acid sequence shown in SEQ ID NO:12, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:13, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:14.

[0148] For example, the isolated antigen-binding protein LCDR1 described in this application may contain the amino acid sequence shown in SEQ ID NO:7, LCDR2 may contain the amino acid sequence shown in SEQ ID NO:15, LCDR3 may contain the amino acid sequence shown in SEQ ID NO:9, and HCDR1 may contain the amino acid sequence shown in SEQ ID NO:12, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:13, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:14.

[0149] FR

[0150] In this application, the VL of the isolated antigen-binding protein may include frame regions L-FR1, L-FR2, L-FR3, and L-FR4.

[0151] In this application, the L-FR1 may include X1X2VX3TQX4X5X6X7X8X9X 10 X 11 X 12 X 13 X 14 X 15 X 16 X 17 X 18 X19X 20 The amino acid sequence shown in SEQ ID NO:38, wherein X1 can be Q or D, X2 can be S or V, X3 can be L or M, X4 can be P or S, X5 can be A or P, X6 can be S or L, X7 can be V or S, X8 can be S or L, X9 can be G or P, X 10 It can be S or V, X 11 It can be P or T, X 12 It can be G or L, X 13 It can be Q or G, X 14 It can be S or Q, X 15 It can be I or P, X 16 It can be T or A, X 17 It can be I or S, X 18 It can be S or I, X 19 It can be C or S, X 20 It can be blank or C.

[0152] For example, the C-terminus of the L-FR1 may be directly or indirectly connected to the N-terminus of the LCDR1, and the L-FR1 may contain the amino acid sequence shown in either SEQ ID NO:22 or 30.

[0153] For example, the isolated antigen-binding protein L-FR1 may contain the amino acid sequence shown in either SEQ ID NO:22 or 30.

[0154] In this application, the L-FR2 may contain the amino acid sequence shown in SEQ ID NO:39.

[0155] WYQQX1PGX2X3PX4LX5IY(SEQ ID NO:39), where X1 can be H or R, X2 can be K or Q, X3 can be A or S, X4 can be K or R, and X5 can be M or L.

[0156] For example, the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 may contain the amino acid sequence shown in either SEQ ID NO: 23 or 31.

[0157] For example, the L-FR2 may contain the amino acid sequence shown in either SEQ ID NO:23 or 31.

[0158] In this application, the L-FR3 may contain the amino acid sequence shown in SEQ ID NO:40.

[0159] GVX1X2RFSGSX3SGX4X5X6X7LX8ISX9X 10 X 11 AEDX 12 X 13 X 14 YYC (SEQ ID NO:40), where X1 can be S or P, X2 can be N or D, X3 can be K or G, X4 can be N or T, X5 can be T or D, X6 can be A or F, X7 can be S or T, X8 can be T or K, X9 can be G or R, X 10 It can be L or V, X 11 It can be Q or E, X 12 It can be E or V, X 13 It can be A or G, X 14 It can be D or V.

[0160] For example, the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 may contain the amino acid sequence shown in either SEQ ID NO: 24 or 32.

[0161] For example, the L-FR3 may contain the amino acid sequence shown in either SEQ ID NO:24 or 32.

[0162] In this application, the L-FR4 may contain the amino acid sequence shown in SEQ ID NO:41.

[0163] FGX1GTKLX2X3X4(SEQ ID NO:41), where X1 can be G or S, X2 can be T or E, X3 can be V or I, and X4 can be L or K.

[0164] For example, the N-terminus of the L-FR4 is connected to the C-terminus of the LCDR3, and the L-FR4 may contain the amino acid sequence shown in either SEQ ID NO: 25 or 33.

[0165] For example, the L-FR4 may contain the amino acid sequence shown in either SEQ ID NO:25 or 33.

[0166] For example, the isolated antigen-binding protein described in this application may contain the amino acid sequence shown in SEQ ID NO:22 for L-FR1, the amino acid sequence shown in SEQ ID NO:23 for L-FR2, the amino acid sequence shown in SEQ ID NO:24 for L-FR3, and the amino acid sequence shown in SEQ ID NO:25 for L-FR4.

[0167] For example, the isolated antigen-binding protein described in this application may contain the amino acid sequence shown in SEQ ID NO:30 for L-FR1, the amino acid sequence shown in SEQ ID NO:31 for L-FR2, the amino acid sequence shown in SEQ ID NO:32 for L-FR3, and the amino acid sequence shown in SEQ ID NO:33 for L-FR4.

[0168] In this application, the VH of the isolated antigen-binding protein may include frame regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0169] In this application, the H-FR1 may contain the amino acid sequence shown in SEQ ID NO:42.

[0170] X1VQLVQSGX2X3X4X5X6PGX7SX8X9X 10 SCX 11 ASGX 12 X 13 FX 14 (SEQ ID NO:42), where X1 can be E or Q, X2 can be G or A, X3 can be G or E, X4 can be L or V, X5 can be V or K, X6 can be Q or K, X7 can be R or A, X8 can be L or V, X9 can be R or K, X 10It can be L or V, X 11 It can be A or K, X 12 It can be F or Y, X 13 It can be T or D, X 14 It can be D or T.

[0171] In this application, the C-terminus of H-FR1 is directly or indirectly connected to the N-terminus of HCDR1, and H-FR1 may contain the amino acid sequence shown in either SEQ ID NO:26 or 34.

[0172] In this application, the H-FR1 may contain the amino acid sequence shown in either SEQ ID NO:26 or 34.

[0173] In this application, the H-FR2 may contain the amino acid sequence shown in SEQ ID NO:43.

[0174] WVRQAPGX1GLEWX2X3(SEQ ID NO:43), where X1 can be K or Q, X2 can be V or M, and X3 can be S or G.

[0175] In this application, H-FR2 is located between HCDR1 and HCDR2, and H-FR2 may contain the amino acid sequence shown in either SEQ ID NO:27 or 35.

[0176] In this application, the H-FR2 may contain the amino acid sequence shown in either SEQ ID NO:27 or 35.

[0177] In this application, the H-FR3 may contain the amino acid sequence shown in SEQ ID NO:44.

[0178] RX1, TX2, X3, X4, DX5, X6, X7, X8, X9 10 YX 11 X 12 X 13 X 14 X 15 LRX 16 X 17 DTX 18 X 19 YYCX 20 X 21 (SEQ ID NO:44), where X1 can be F or V, X2 can be I or L, X3 can be S or T, X4 can be R or A, X5 can be N or K, X6 can be A or S, X7 can be K or I, X8 can be N or S, X9 can be S or T, X 10 It can be L or A, X11 It can be L or M, X 12 It can be Q or E, X 13 It can be M or L, X 14 It can be N or S, X 15 It can be S or R, X 16 It can be A or S, X 17 It can be E or D, X 18 It can be A or V, X 19 It can be L or V, X 20 It can be A or blank, X 21 It can be K or blank.

[0179] In this application, H-FR3 is located between HCDR2 and HCDR3, and H-FR3 may contain the amino acid sequence shown in either SEQ ID NO:28 or 36.

[0180] In this application, the H-FR3 may contain the amino acid sequence shown in either SEQ ID NO:28 or 36.

[0181] In this application, the H-FR4 may contain the amino acid sequence shown in SEQ ID NO:45.

[0182] WGQGTX1VTVSS (SEQ ID NO:45), where X1 can be M or L.

[0183] In this application, the N-terminus of H-FR4 is connected to the C-terminus of HCDR3, and H-FR4 may contain the amino acid sequence shown in either SEQ ID NO:29 or 37.

[0184] In this application, the H-FR4 may contain the amino acid sequence shown in either SEQ ID NO:29 or 37.

[0185] For example, the isolated antigen-binding protein described in this application may contain the amino acid sequence shown in SEQ ID NO:26 for H-FR1, the amino acid sequence shown in SEQ ID NO:27 for H-FR2, the amino acid sequence shown in SEQ ID NO:28 for H-FR3, and the amino acid sequence shown in SEQ ID NO:29 for H-FR4.

[0186] For example, the isolated antigen-binding protein described in this application may contain the amino acid sequence shown in SEQ ID NO:34 for H-FR1, the amino acid sequence shown in SEQ ID NO:35 for H-FR2, the amino acid sequence shown in SEQ ID NO:36 for H-FR3, and the amino acid sequence shown in SEQ ID NO:37 for H-FR4.

[0187] For example, the isolated antigen-binding protein described in this application may contain the amino acid sequence shown in SEQ ID NO:22 for L-FR1, the amino acid sequence shown in SEQ ID NO:23 for L-FR2, the amino acid sequence shown in SEQ ID NO:24 for L-FR3, the amino acid sequence shown in SEQ ID NO:25 for L-FR4, and the amino acid sequence shown in SEQ ID NO:26 for H-FR1, the amino acid sequence shown in SEQ ID NO:27 for H-FR2, the amino acid sequence shown in SEQ ID NO:28 for H-FR3, and the amino acid sequence shown in SEQ ID NO:29 for H-FR4.

[0188] For example, the isolated antigen-binding protein described in this application may contain the amino acid sequence shown in SEQ ID NO:30 for L-FR1, the amino acid sequence shown in SEQ ID NO:31 for L-FR2, the amino acid sequence shown in SEQ ID NO:32 for L-FR3, and the amino acid sequence shown in SEQ ID NO:33 for L-FR4. Furthermore, H-FR1 may contain the amino acid sequence shown in SEQ ID NO:34 for H-FR2, the amino acid sequence shown in SEQ ID NO:35 for H-FR3, the amino acid sequence shown in SEQ ID NO:36 for H-FR4, and the amino acid sequence shown in SEQ ID NO:37 for H-FR4.

[0189] VL and VH

[0190] The isolated antigen-binding protein described in this application may comprise an antibody light chain variable region (VL) and an antibody heavy chain variable region (VH). For example, the VL may comprise the amino acid sequence shown in SEQ ID NO:49, and the VH may comprise the amino acid sequence shown in SEQ ID NO:53. As another example, the VL of the isolated antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:46-48. The VH of the isolated antigen-binding protein may comprise the amino acid sequence shown in any one of SEQ ID NO:50-52.

[0191] For example, the VL may contain the amino acid sequence shown in SEQ ID NO:46, and the VH may contain the amino acid sequence shown in SEQ ID NO:50.

[0192] For example, the VL may contain the amino acid sequence shown in SEQ ID NO:47, and the VH may contain the amino acid sequence shown in SEQ ID NO:51.

[0193] For example, the VL may contain the amino acid sequence shown in SEQ ID NO:47, and the VH may contain the amino acid sequence shown in SEQ ID NO:52.

[0194] For example, the VL may contain the amino acid sequence shown in SEQ ID NO:48, and the VH may contain the amino acid sequence shown in SEQ ID NO:52.

[0195] Light chains and heavy chains

[0196] In this application, the isolated antigen-binding protein may include an antibody light chain constant region, and the antibody light chain constant region may include a human Igκ constant region. For example, the antibody light chain constant region may contain the amino acid sequence shown in SEQ ID NO:54.

[0197] In this application, the isolated antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from the human IgG heavy chain constant region. In some embodiments, the isolated antigen-binding protein may include an antibody heavy chain constant region, and the antibody heavy chain constant region may be derived from the human IgG1 heavy chain constant region. For example, the antibody heavy chain constant region may contain the amino acid sequence shown in SEQ ID NO:55.

[0198] In this application, the isolated antigen-binding protein may comprise an antibody light chain LC, and the LC may comprise an amino acid sequence represented by any one of SEQ ID NO:56-58.

[0199] In this application, the isolated antigen-binding protein may contain an antibody heavy chain HC, and the HC may contain the amino acid sequence shown in any one of SEQ ID NO:59-61.

[0200] The isolated antigen-binding protein described in this application may comprise antibody light chains and antibody heavy chains.

[0201] For example, the light chain may contain the amino acid sequence shown in SEQ ID NO:56, and the heavy chain may contain the amino acid sequence shown in SEQ ID NO:59.

[0202] For example, the light chain may contain the amino acid sequence shown in SEQ ID NO:57, and the heavy chain may contain the amino acid sequence shown in SEQ ID NO:60.

[0203] For example, the light chain may contain the amino acid sequence shown in SEQ ID NO:57, and the heavy chain may contain the amino acid sequence shown in SEQ ID NO:61.

[0204] For example, the light chain may contain the amino acid sequence shown in SEQ ID NO:58, and the heavy chain may contain the amino acid sequence shown in SEQ ID NO:61.

[0205] In this application, the light chain of the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:56, and the heavy chain may contain the amino acid sequence shown in SEQ ID NO:59. Specifically, LCDR1 of the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:1, LCDR2 may contain the amino acid sequence shown in SEQ ID NO:2, LCDR3 may contain the amino acid sequence shown in SEQ ID NO:3, and HCDR1 may contain the amino acid sequence shown in SEQ ID NO:4, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:5, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:6. The isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:22 for L-FR1, the amino acid sequence shown in SEQ ID NO:23 for L-FR2, the amino acid sequence shown in SEQ ID NO:24 for L-FR3, and the amino acid sequence shown in SEQ ID NO:25 for L-FR4. Furthermore, H-FR1 may contain the amino acid sequence shown in SEQ ID NO:26, H-FR2 may contain the amino acid sequence shown in SEQ ID NO:27, H-FR3 may contain the amino acid sequence shown in SEQ ID NO:28, and H-FR4 may contain the amino acid sequence shown in SEQ ID NO:29. The VL may contain the amino acid sequence shown in SEQ ID NO:46, and the VH may contain the amino acid sequence shown in SEQ ID NO:50. For example, the isolated antigen-binding protein may be 204A.

[0206] In this application, the light chain of the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:57, and the heavy chain may contain the amino acid sequence shown in SEQ ID NO:60. Specifically, LCDR1 of the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:7, LCDR2 may contain the amino acid sequence shown in SEQ ID NO:8, LCDR3 may contain the amino acid sequence shown in SEQ ID NO:9, and HCDR1 may contain the amino acid sequence shown in SEQ ID NO:12, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:10, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:11. The isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:30 for L-FR1, SEQ ID NO:31 for L-FR2, SEQ ID NO:32 for L-FR3, and SEQ ID NO:33 for L-FR4. Furthermore, H-FR1 may contain the amino acid sequence shown in SEQ ID NO:34, H-FR2 may contain the amino acid sequence shown in SEQ ID NO:35, H-FR3 may contain the amino acid sequence shown in SEQ ID NO:36, and H-FR4 may contain the amino acid sequence shown in SEQ ID NO:37. The VL may contain the amino acid sequence shown in SEQ ID NO:47, and the VH may contain the amino acid sequence shown in SEQ ID NO:51. For example, the isolated antigen-binding protein may be L1H2.

[0207] In this application, the light chain of the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:57, and the heavy chain may contain the amino acid sequence shown in SEQ ID NO:61. Specifically, LCDR1 of the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:7, LCDR2 may contain the amino acid sequence shown in SEQ ID NO:8, LCDR3 may contain the amino acid sequence shown in SEQ ID NO:9, and HCDR1 may contain the amino acid sequence shown in SEQ ID NO:12, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:13, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:14. The isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:30 for L-FR1, SEQ ID NO:31 for L-FR2, SEQ ID NO:32 for L-FR3, and SEQ ID NO:33 for L-FR4. Furthermore, H-FR1 may contain the amino acid sequence shown in SEQ ID NO:34, H-FR2 may contain the amino acid sequence shown in SEQ ID NO:35, H-FR3 may contain the amino acid sequence shown in SEQ ID NO:36, and H-FR4 may contain the amino acid sequence shown in SEQ ID NO:37. The VL may contain the amino acid sequence shown in SEQ ID NO:47, and the VH may contain the amino acid sequence shown in SEQ ID NO:52. For example, the isolated antigen-binding protein may be L1H6.

[0208] In this application, the light chain of the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:58, and the heavy chain may contain the amino acid sequence shown in SEQ ID NO:61. Specifically, LCDR1 of the isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:7, LCDR2 may contain the amino acid sequence shown in SEQ ID NO:15, LCDR3 may contain the amino acid sequence shown in SEQ ID NO:9, and HCDR1 may contain the amino acid sequence shown in SEQ ID NO:12, HCDR2 may contain the amino acid sequence shown in SEQ ID NO:13, and HCDR3 may contain the amino acid sequence shown in SEQ ID NO:14. The isolated antigen-binding protein may contain the amino acid sequence shown in SEQ ID NO:30 for L-FR1, SEQ ID NO:31 for L-FR2, SEQ ID NO:32 for L-FR3, and SEQ ID NO:33 for L-FR4. Furthermore, H-FR1 may contain the amino acid sequence shown in SEQ ID NO:34, H-FR2 may contain the amino acid sequence shown in SEQ ID NO:35, H-FR3 may contain the amino acid sequence shown in SEQ ID NO:36, and H-FR4 may contain the amino acid sequence shown in SEQ ID NO:37. VL may contain the amino acid sequence shown in SEQ ID NO:48, and VH may contain the amino acid sequence shown in SEQ ID NO:52. For example, the isolated antigen-binding protein may be L2H6.

[0209] Nucleic acid molecules, vectors, cells, preparation methods, and pharmaceutical compositions

[0210] On the other hand, this application also provides one or more isolated nucleic acid molecules that can encode the isolated antigen-binding protein described in this application. The one or more isolated nucleic acid molecules described in this application can be nucleotides, deoxyribonucleotides, or ribonucleotides of any length in isolated form, or analogs isolated from their natural environment or artificially synthesized, but can encode the isolated antigen-binding protein described in this application.

[0211] On the other hand, this application also provides a vector that may contain the nucleic acid molecules described in this application. The vector can be transformed, transduced, or transfected into host cells, enabling the expression of its carried genetic material elements within the host cells. For example, the vector may include: plasmids; phage particles; Cos plasmids; artificial chromosomes such as yeast artificial chromosomes (YAC), bacterial artificial chromosomes (BAC), or P1-derived artificial chromosomes (PAC); bacteriophages such as λ phage or M13 phage; and animal viruses, etc. Animal viruses used as vectors include retrotranscriptoviruses (including lentiviruses), adenoviruses, adeno-associated viruses, herpesviruses (such as herpes simplex virus), poxviruses, baculoviruses, papillomaviruses, and papillomaviruses (such as SV40). For example, the vector may contain various elements controlling expression, including promoter sequences, transcription initiation sequences, enhancer sequences, selection elements, and reporter genes. Additionally, the vector may contain a replication initiation site. Furthermore, the vector may include components that facilitate its entry into the cell, such as viral particles, liposomes, or protein coats, but not only these substances.

[0212] On the other hand, this application also provides cells that may contain the nucleic acid molecules or vectors described in this application. The cells may include progeny of a single cell. Due to natural, accidental, or intentional mutations, the progeny may not necessarily be identical to the original parent cell (in the morphology of the total DNA complement or in the genome). In some embodiments, the cells may also include cells transfected in vitro using the vectors described in this invention. In some embodiments, the cells may be bacterial cells (e.g., *E. coli*), yeast cells, or other eukaryotic cells, such as COS cells, Chinese hamster ovary (CHO) cells, HeLa cells, HEK293 cells, COS-1 cells, NSO cells, or myeloma cells. In some embodiments, the cells may be mammalian cells. In some embodiments, the mammalian cells may be HEK293 cells.

[0213] On the other hand, this application also provides a method for preparing the isolated antigen-binding protein described in this application, the method including culturing the cells described in this application under conditions that cause the isolated antigen-binding protein described in this application to be expressed.

[0214] On the other hand, this application also provides pharmaceutical compositions that may comprise the isolated antigen-binding protein described in this application, the nucleic acid molecule described in this application, the carrier described in this application, and / or the cell described in this application, and optionally a pharmaceutically acceptable adjuvant.

[0215] In some embodiments, the pharmaceutical composition may further comprise suitable formulations of one or more (pharmaceutically effective) carriers, stabilizers, excipients, diluents, solubilizers, surfactants, emulsifiers, and / or preservatives. The acceptable components of the composition are preferably non-toxic to the recipient at the dosage and concentration used. The pharmaceutical compositions of the present invention include, but are not limited to, liquid, freeze-dried, and lyophilized compositions.

[0216] In some embodiments, the pharmaceutically acceptable adjuvant may include any and all solvents, dispersion media, coatings, isotonic agents and absorption delay agents that are compatible with drug administration, are generally safe and non-toxic, and are neither biologically nor otherwise undesirable.

[0217] In some embodiments, the pharmaceutical composition may be administered parenterally, percutaneously, intracavitarily, intra-arterially, intrathecally, and / or intranasally, or directly injected into tissues. For example, the pharmaceutical composition may be administered to a patient or subject by infusion or injection. In some embodiments, the pharmaceutical composition may be administered in various ways, such as intravenously, intraperitoneally, subcutaneously, intramuscularly, locally, or intradermally. In some embodiments, the pharmaceutical composition may be administered continuously. This continuous (or uninterrupted) administration may be achieved using a small pump system worn by the patient to measure the amount of therapeutic agent flowing into the patient, as described in WO2015 / 036583.

[0218] Uses and applications

[0219] On the other hand, this application also provides the use of the isolated antigen-binding protein described in this application, the nucleic acid molecule described in this application, the carrier described in this application, the cell described in this application, and / or the pharmaceutical composition described in this application in the preparation of a medicament that can be used to prevent, alleviate, and / or treat tumors.

[0220] On the other hand, this application also provides methods for preventing, alleviating, or treating tumors, which may include administering the isolated antigen-binding protein described in this application to a subject in need. In this application, the administration may be performed in various ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, local, or intradermal administration.

[0221] On the other hand, the isolated antigen-binding protein, the nucleic acid molecule, the carrier, the cell, and / or the pharmaceutical composition described in this application can be used to prevent, alleviate, or treat tumors.

[0222] In this application, the tumor can be a solid tumor or a hematologic malignancy. For example, the tumor can include a GPC3-positive tumor, which can include liver cancer.

[0223] In this application, the subject may include humans and non-human animals. For example, the subject may include, but is not limited to, cats, dogs, horses, pigs, cows, sheep, rabbits, mice, rats, or monkeys.

[0224] On the other hand, this application also provides a method for detecting GPC3 in a sample, the method comprising administering the isolated antigen-binding protein described in this application. In this application, the administration can be performed in various ways, such as intravenous, intratumoral, intraperitoneal, subcutaneous, intramuscular, local, or intradermal administration.

[0225] The embodiments described below are not intended to be limited by any theory, but are merely for illustrating the protein molecules, preparation methods, and uses of this application, and are not intended to limit the scope of the invention. The embodiments do not include detailed descriptions of conventional methods, such as those used to construct vectors and plasmids, methods for inserting genes encoding proteins into such vectors and plasmids, or methods for introducing plasmids into host cells. Such methods are well known to those skilled in the art and have been described in numerous publications, including Sambrook, J., Fritsch, E.F. and Maniais, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press.

[0226] Implementation Plan

[0227] 11. An isolated antigen-binding protein containing at least one CDR of the VH amino acid sequence as shown in SEQ ID NO:53; and containing at least one CDR of the VL amino acid sequence as shown in SEQ ID NO:49.

[0228] 12. The antigen-binding protein isolated according to embodiment 1 has one or more of the following properties:

[0229] 1) Able to use 6×10 -9 M or lower K D Binds to the GPC3 protein, wherein the K D The value was determined by Octet;

[0230] 2) In FACS assays, it can specifically bind to the GPC3 protein on the surface of HepG2 cells and / or Huh7 cells;

[0231] 3) It can inhibit tumor growth and / or tumor cell proliferation.

[0232] 13. The isolated antigen-binding protein according to embodiment 2, wherein the GPC3 protein comprises human GPC3 protein.

[0233] 14. An isolated antigen-binding protein according to any one of embodiments 2-3, wherein the human GPC3 protein comprises the amino acid sequence shown in SEQ ID NO:74.

[0234] 15. An antigen-binding protein isolated according to any one of embodiments 2-4, wherein the tumor comprises a GPC3-positive tumor.

[0235] 16. The isolated antigen-binding protein according to embodiment 5, wherein the GPC3-positive tumor includes liver cancer.

[0236] 17 An isolated antigen-binding protein according to any one of embodiments 1-6, comprising HCDR1 in VH as shown in SEQ ID NO:53.

[0237] 18 An isolated antigen-binding protein according to any one of embodiments 1-7, comprising HCDR2 in VH as shown in SEQ ID NO:53.

[0238] 19 An isolated antigen-binding protein according to any one of embodiments 1-8, comprising HCDR3 in VH as shown in SEQ ID NO:53.

[0239] 20. An isolated antigen-binding protein according to embodiment 7, wherein HCDR1 comprises the amino acid sequence shown in X1YX2MH, wherein X1 can be D or A, and X2 can be A or E.

[0240] 21. An isolated antigen-binding protein according to embodiment 10, wherein the HCDR1 comprises the amino acid sequence shown in either SEQ ID NO:4 or 12.

[0241] 22. An isolated antigen-binding protein according to embodiment 8, wherein the HCDR2 comprises the amino acid sequence shown in SEQ ID NO:20.

[0242] 23. An isolated antigen-binding protein according to embodiment 12, wherein the HCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 5, 10 and 13.

[0243] 24. The isolated antigen-binding protein according to embodiment 9, wherein the HCDR3 comprises X1X2X3X4X5X6X7X8X9X 10 X 11The amino acid sequence shown can be D or T, X2 can be H or R, X3 can be T or F, X4 can be I or Y, X5 can be G or S, X6 can be V or Y, X7 can be G or A, X8 can be A, Y or H, X9 can be F or blank, X 10 Can be D or blank, X 11 It can be 'I' or blank.

[0244] 25 An isolated antigen-binding protein according to embodiment 14, wherein the HCDR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 6, 11 and 14.

[0245] 26 An isolated antigen-binding protein according to any one of embodiments 1-15, comprising LCDR1 in VL as shown in SEQ ID NO:49.

[0246] 27 An isolated antigen-binding protein according to any one of embodiments 1-16, comprising LCDR2 in VL as shown in SEQ ID NO:49.

[0247] 28 An isolated antigen-binding protein according to any one of embodiments 1-17, comprising LCDR3 in VL as shown in SEQ ID NO:49.

[0248] 29. The isolated antigen-binding protein according to embodiment 16, wherein the LCDR1 comprises X1X2X3X4SX5VX6X7X8X9YX 10 X 11 X 12 X 13 The amino acid sequence shown can be T or R, X2 can be G or S, X3 can be T or S, X4 can be S or Q, X5 can be D or L, X6 can be G or H, X7 can be G or S, X8 can be Y or N, X9 can be N or G, X... 10 It can be V or T, X 11 It can be S or Y, X 12 Can be blank or L, X 13 It can be blank or H.

[0249] 30. An isolated antigen-binding protein according to embodiment 19, wherein the LCDR1 comprises the amino acid sequence shown in either SEQ ID NO:1 or 7.

[0250] 31. The isolated antigen-binding protein according to embodiment 17, wherein the LCDR2 comprises the amino acid sequence shown in X1X2SX3RX4S, wherein X1 can be D or K, X2 can be V or G, X3 can be N, Y or Q, and X4 can be P or G.

[0251] 32. An isolated antigen-binding protein according to embodiment 21, wherein the LCDR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 2, 8 and 15.

[0252] 33. The isolated antigen-binding protein according to embodiment 18, wherein the LCDR3 comprises X1X2X3X4X5X6X7X8X9X 10 The amino acid sequence shown can be S or G, X2 can be S or Q, X3 can be Y or S, X4 can be A or G, X5 can be S or L, X6 can be G or T, X7 can be S or P, X8 can be T or P, X9 can be L or T, X... 10 It can be a V or a blank.

[0253] 34. An isolated antigen-binding protein according to embodiment 23, wherein the LCDR3 comprises the amino acid sequence shown in either SEQ ID NO:3 or 9.

[0254] 35 An isolated antigen-binding protein according to any one of embodiments 1-24, comprising an antibody or an antigen-binding fragment thereof.

[0255] 36. An isolated antigen-binding protein according to embodiment 25, wherein the antigen-binding fragment includes Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and / or dAb.

[0256] 37 An isolated antigen-binding protein according to any one of embodiments 1-26, wherein the VL includes frame regions L-FR1, L-FR2, L-FR3, and L-FR4.

[0257] 38. An isolated antigen-binding protein according to embodiment 27, wherein the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1, and the L-FR1 comprises the amino acid sequence shown in either SEQ ID NO:22 or 30.

[0258] 39 An isolated antigen-binding protein according to any one of embodiments 27-28, wherein the L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:22 and 30.

[0259] 40. An isolated antigen-binding protein according to embodiment 27, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence shown in either SEQ ID NO: 23 or 31.

[0260] 41 An isolated antigen-binding protein according to any one of embodiments 27 and 30, wherein the L-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 23 and 31.

[0261] 42. An isolated antigen-binding protein according to embodiment 27, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence shown in either SEQ ID NO:24 or 32.

[0262] 43 An isolated antigen-binding protein according to any one of embodiments 27 and 32, wherein the L-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 24 and 32.

[0263] 44. An isolated antigen-binding protein according to embodiment 27, wherein the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence shown in either SEQ ID NO:25 or 33.

[0264] 45 An isolated antigen-binding protein according to any one of embodiments 27 and 34, wherein the L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO: 25 and 33.

[0265] 46 An isolated antigen-binding protein according to any one of embodiments 1-35, wherein the VL comprises the amino acid sequence shown in any one of SEQ ID NO:46-48.

[0266] 47. An isolated antigen-binding protein according to any one of embodiments 1-36, comprising an antibody light chain constant region, wherein the antibody light chain constant region comprises a human Igκ constant region.

[0267] 48. An isolated antigen-binding protein according to embodiment 37, wherein the constant region of the antibody light chain contains the amino acid sequence shown in SEQ ID NO:54.

[0268] 49 An isolated antigen-binding protein according to any one of embodiments 1-38, comprising an antibody light chain LC, wherein the LC comprises an amino acid sequence shown in any one of SEQ ID NO:56-58.

[0269] 50 An isolated antigen-binding protein according to any one of embodiments 1-39, wherein the VH includes frame regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0270] 51. An isolated antigen-binding protein according to embodiment 40, wherein the C-terminus of H-FR1 is directly or indirectly linked to the N-terminus of HCDR1, and the H-FR1 comprises the amino acid sequence shown in either SEQ ID NO: 26 or 34.

[0271] 52 An isolated antigen-binding protein according to any one of embodiments 40-41, wherein the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO:26 and 34.

[0272] 53 An isolated antigen-binding protein according to embodiment 40, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 27 and 35.

[0273] 54 An isolated antigen-binding protein according to any one of embodiments 40 and 43, wherein the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 27 and 35.

[0274] 55. An isolated antigen-binding protein according to embodiment 40, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in either SEQ ID NO:28 or 36.

[0275] 56 An isolated antigen-binding protein according to any one of embodiments 40 and 45, wherein the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 28 and 36.

[0276] 57 An isolated antigen-binding protein according to embodiment 40, wherein the N-terminus of H-FR4 is linked to the C-terminus of HCDR3, and the H-FR4 comprises the amino acid sequence shown in either SEQ ID NO:29 or 37.

[0277] 58 An isolated antigen-binding protein according to any one of embodiments 40 and 47, wherein the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO: 29 and 37.

[0278] 59 An isolated antigen-binding protein according to any one of embodiments 1-48, wherein the VH comprises the amino acid sequence shown in any one of SEQ ID NO:50-52.

[0279] 60. An isolated antigen-binding protein according to any one of embodiments 1-49, comprising an antibody heavy chain constant region, wherein the antibody heavy chain constant region is derived from the human IgG heavy chain constant region.

[0280] 61. An isolated antigen-binding protein according to any one of embodiments 1-50, comprising an antibody heavy chain constant region, wherein the antibody heavy chain constant region is derived from the human IgG1 heavy chain constant region.

[0281] 62 An isolated antigen-binding protein according to any one of embodiments 1-51, wherein the constant region of the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO:55.

[0282] 63 An isolated antigen-binding protein according to any one of embodiments 1-52, comprising an antibody heavy chain HC, wherein the HC comprises an amino acid sequence shown in any one of SEQ ID NO:59-61.

[0283] 64. One or more isolated nucleic acid molecules that encode the isolated antigen-binding protein as described in any one of embodiments 1-53.

[0284] 65. A vector containing nucleic acid molecules as described in embodiment 54.

[0285] 66 cells, which contain nucleic acid molecules according to embodiment 54 or vectors according to embodiment 55.

[0286] 67. A method for preparing the isolated antigen-binding protein of any one of embodiments 1-53, the method comprising culturing cells according to embodiment 56 under conditions that cause expression of the isolated antigen-binding protein of any one of embodiments 1-53.

[0287] 68. A pharmaceutical composition comprising, as described in any one of embodiments 1-53, a nucleic acid molecule as described in embodiment 54, a carrier as described in embodiment 55 and / or a cell as described in embodiment 56, and optionally a pharmaceutically acceptable adjuvant.

[0288] 69. Use of the isolated antigen-binding protein of any one of embodiments 1-53, the nucleic acid molecule of embodiment 54, the carrier of embodiment 55, the cell of embodiment 56, and / or the pharmaceutical composition of embodiment 58 in the preparation of a medicament for the prevention, relief, and / or treatment of tumors.

[0289] 70. A method for preventing, alleviating, or treating tumors, said method comprising administering to a subject in need the isolated antigen-binding protein described in any one of embodiments 1-53.

[0290] 71. A method for detecting GPC3 in a sample, the method comprising administering the isolated antigen-binding protein as described in any one of embodiments 1-53.

[0291] This application also provides the following implementation methods:

[0292] 1. An isolated antigen-binding protein capable of binding to a GPC3 protein, said antigen-binding protein comprising an antibody light chain or a fragment thereof and an antibody heavy chain or a fragment thereof, said antibody light chain or fragment thereof comprising LCDR1, LCDR2, and LCDR3, said antibody heavy chain or fragment thereof comprising HCDR1, HCDR2, and HCDR3, said LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 comprising an amino acid sequence selected from the group consisting of:

[0293] (1) HCDR1: SEQ ID NO: 12, HCDR2: SEQ ID NO: 13, HCDR3: SEQ ID NO: 14, LCDR1: SEQ ID NO: 7, LCDR2: SEQ ID NO: 8, LCDR3: SEQ ID NO: 9;

[0294] (2) HCDR1: SEQ ID NO: 12, HCDR2: SEQ ID NO: 13, HCDR3: SEQ ID NO: 14, LCDR1: SEQ ID NO: 7, LCDR2: SEQ ID NO: 15, LCDR3: SEQ ID NO: 9; and

[0295] (3) HCDR1: SEQ ID NO:4, HCDR2: SEQ ID NO:5, HCDR3: SEQ ID NO:6, LCDR1: SEQ ID NO:1, LCDR2: SEQ ID NO:2, LCDR3: SEQ ID NO:3.

[0296] 2. The antigen-binding protein isolated according to Embodiment 1 has one or more of the following properties:

[0297] 1) Able to use 6×10 -9 M or lower K D Binds to the GPC3 protein, wherein the K D The value was determined by Octet;

[0298] 2) In FACS assays, it can specifically bind to the GPC3 protein on the surface of HepG2 cells and / or Huh7 cells;

[0299] 3) It can inhibit tumor growth and / or tumor cell proliferation.

[0300] 3. The isolated antigen-binding protein according to Embodiment 2, wherein the GPC3 protein comprises human GPC3 protein.

[0301] 4. The isolated antigen-binding protein according to any one of embodiments 2-3, wherein the human GPC3 protein comprises the amino acid sequence shown in SEQ ID NO:74.

[0302] 5. The isolated antigen-binding protein according to any one of embodiments 2-4, wherein the tumor comprises a GPC3-positive tumor.

[0303] 6. The isolated antigen-binding protein according to Embodiment 5, wherein the GPC3-positive tumor includes liver cancer.

[0304] 7. The isolated antigen-binding protein according to any one of embodiments 1-6, comprising an antibody or an antigen-binding fragment thereof.

[0305] 8. The isolated antigen-binding protein according to Embodiment 7, wherein the antigen-binding fragments include Fab, Fab', F(ab)2, Fv fragment, F(ab')2, scFv, di-scFv and / or dAb.

[0306] 9. The isolated antigen-binding protein according to any one of embodiments 1-8, wherein the antibody light chain or a fragment thereof comprises frame regions L-FR1, L-FR2, L-FR3, and L-FR4.

[0307] 10. The isolated antigen-binding protein according to Embodiment 9, wherein the C-terminus of the L-FR1 is directly or indirectly linked to the N-terminus of the LCDR1, and the L-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 22 and 30.

[0308] 11. The isolated antigen-binding protein according to any one of embodiments 9-10, wherein the L-FR2 is located between the LCDR1 and the LCDR2, and the L-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 23 and 31.

[0309] 12. The isolated antigen-binding protein according to any one of embodiments 9-11, wherein the L-FR3 is located between the LCDR2 and the LCDR3, and the L-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 24 and 32.

[0310] 13. The isolated antigen-binding protein according to any one of embodiments 9-12, wherein the N-terminus of the L-FR4 is linked to the C-terminus of the LCDR3, and the L-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO: 25 and 33.

[0311] 14. The isolated antigen-binding protein according to any one of embodiments 1-13, wherein the antibody light chain or a fragment thereof comprises a light chain variable region VL, and the VL comprises an amino acid sequence shown in any one of SEQ ID NO:46-48.

[0312] 15. The isolated antigen-binding protein according to any one of embodiments 1-14, comprising an antibody light chain constant region, wherein the antibody light chain constant region comprises a human Igκ constant region.

[0313] 16. The isolated antigen-binding protein according to embodiment 15, wherein the constant region of the antibody light chain comprises the amino acid sequence shown in SEQ ID NO:54.

[0314] 17. The isolated antigen-binding protein according to any one of embodiments 1-16, wherein the antibody light chain comprises the amino acid sequence shown in any one of SEQ ID NO:56-58.

[0315] 18. The isolated antigen-binding protein according to any one of embodiments 1-17, wherein the antibody heavy chain or a fragment thereof comprises frame regions H-FR1, H-FR2, H-FR3, and H-FR4.

[0316] 19. The isolated antigen-binding protein according to embodiment 18, wherein the C-terminus of H-FR1 is directly or indirectly linked to the N-terminus of HCDR1, and the H-FR1 comprises the amino acid sequence shown in any one of SEQ ID NO: 26 and 34.

[0317] 20. The isolated antigen-binding protein according to any one of embodiments 18-19, wherein the H-FR2 is located between the HCDR1 and the HCDR2, and the H-FR2 comprises the amino acid sequence shown in any one of SEQ ID NO: 27 and 35.

[0318] 21. The isolated antigen-binding protein according to any one of embodiments 18-20, wherein the H-FR3 is located between the HCDR2 and the HCDR3, and the H-FR3 comprises the amino acid sequence shown in any one of SEQ ID NO: 28 and 36.

[0319] 22. The isolated antigen-binding protein according to any one of embodiments 18-21, wherein the N-terminus of the H-FR4 is linked to the C-terminus of the HCDR3, and the H-FR4 comprises the amino acid sequence shown in any one of SEQ ID NO: 29 and 37.

[0320] 23. The isolated antigen-binding protein according to any one of embodiments 1-22, wherein the antibody heavy chain or a fragment thereof comprises a heavy chain variable region VH, the VH comprising the amino acid sequence shown in SEQ ID NO: 50 or 52.

[0321] 24. The isolated antigen-binding protein according to any one of embodiments 1-23, comprising an antibody heavy chain constant region, wherein the antibody heavy chain constant region is derived from the human IgG heavy chain constant region.

[0322] 25. The isolated antigen-binding protein according to any one of embodiments 1-24, comprising an antibody heavy chain constant region, wherein the antibody heavy chain constant region is derived from the human IgG1 heavy chain constant region.

[0323] 26. The isolated antigen-binding protein according to any one of embodiments 24-25, wherein the constant region of the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO:55.

[0324] 27. The isolated antigen-binding protein according to any one of embodiments 1-26, wherein the antibody heavy chain comprises the amino acid sequence shown in SEQ ID NO:59 or 61.

[0325] 28. One or more isolated nucleic acid molecules that encode the isolated antigen-binding protein of any one of embodiments 1-27.

[0326] 29. A carrier comprising the nucleic acid molecule according to embodiment 28.

[0327] 30. A cell comprising a nucleic acid molecule according to embodiment 28 or a carrier according to embodiment 29.

[0328] 31. A method for preparing the isolated antigen-binding protein of any one of Embodiments 1-27, the method comprising culturing cells according to Embodiment 30 under conditions that cause expression of the isolated antigen-binding protein of any one of Embodiments 1-27.

[0329] 32. A pharmaceutical composition comprising the isolated antigen-binding protein of any one of embodiments 1-27, the nucleic acid molecule of embodiment 28, the carrier of embodiment 29 and / or the cell of embodiment 30, and optionally a pharmaceutically acceptable adjuvant.

[0330] 33. Use of the isolated antigen-binding protein of any one of Embodiments 1-27, the nucleic acid molecule of Embodiment 28, the carrier of Embodiment 29, the cell of Embodiment 30, and / or the pharmaceutical composition of Embodiment 32 in the preparation of a medicament for the prevention, relief, and / or treatment of tumors.

[0331] 34. A method for detecting GPC3 in a sample, the method comprising administering the isolated antigen-binding protein as described in any one of embodiments 1-27.

[0332] Example

[0333] Example 1. Screening for anti-GPC3 antibodies using a phage antibody library

[0334] Using recombinant human GPC3 protein (Shanghai Yuaneng Cell Medical Technology Co., Ltd., amino acid sequence as shown in SEQ ID NO:74) as the antigen, a phage natural human antibody library (Shanghai Yuaneng Cell Medical Technology Co., Ltd.) was sorted in four rounds. 1 ml of GPC3 antigen was coated in CBS buffer in an ELISA tube at a concentration of 20 μg / ml (first and second rounds) or 10 μg / ml (third and fourth rounds), and incubated overnight at 4°C. The next day, the tube was blocked with 2 ml of PBS buffer containing 10% skim milk powder. 1 ml of blocked phage was added to the tube and incubated at room temperature for 1 hour. The tube was washed 10 times (first and second rounds) or 15 times (third and fourth rounds) with PBST. 800 μl of pH 2.2 Gly-HCl buffer was added for elution, and 400 μl of pH 8.0 Tris-HCl buffer was immediately added for neutralization. 20 ml of E. coli strain in the logarithmic growth phase (OD value approximately 0.8) was added. In SS320, mix well and let stand at 37°C for 1 hour; take out 500 μl to determine the phage titer and preserve the bacteria with glycerol; plate the remaining bacterial solution and incubate overnight at 37°C; scrape off the bacteria from the plate the next day and inoculate them into 80 ml of 2YT-Amp medium at a certain ratio to make the OD equal to 0.2. Incubate for several hours until the OD reaches 0.8, then add 160 μl of helper phage, mix well and let stand at 37°C for 1 hour; add isopropyl thiogalactoside (IPTG) and Kan antibiotic, and incubate overnight at 30°C with shaking at 250 rpm; collect the supernatant, precipitate the phage with PEG / NaCl solution, and resuspend the precipitated phage in 1.5 ml of PBS buffer; the resuspended phage is used for the next round of enrichment screening. After 4 rounds of washing, significant enrichment was observed. The panned phage antibody clones were identified using ELISA. The specific method was as follows: Human GPC3 protein (Shanghai Yuaneng Cell Medical Technology Co., Ltd.) was coated onto a 96-well ELISA plate at a concentration of 1 μg / ml and incubated overnight at 4°C. Non-specific binding sites were then blocked with 10% skim milk powder. After thorough washing, the supernatant from a single phage clone was added to the 96-well plate and incubated at 37°C for 2 hours. After thorough washing, Anti-M13-HPR (GE Healtcare, 27-9421-01) was added and incubated at 37°C for 45 minutes. After thorough washing, TMB was added for color development, and the reaction was incubated at room temperature for 5-10 minutes. Finally, the reaction was terminated with sulfuric acid, and the OD value of each well was measured at 450 nm.

[0335] Four phage antibody clones that specifically bind to human GPC3 (i.e., the antigen-binding proteins isolated in this application) were obtained by ELISA identification. Sequencing yielded the VH and VL gene sequences, and these four phage antibody clones were named 204A, L1H2, L1H6, and L2H6, respectively. SEQ ID NO:62 and SEQ ID NO:46 show the nucleic acid and amino acid sequences of the light chain variable region VL of phage antibody clone 204A, respectively. SEQ ID NO:65 and SEQ ID NO:50 show the nucleic acid and amino acid sequences of the heavy chain variable region VH of phage antibody clone 204A, respectively. SEQ ID NO:63 and SEQ ID NO:47 show the nucleic acid and amino acid sequences of the light chain variable region VL of phage antibody clone L1H2, respectively. SEQ ID NO:66 and SEQ ID NO:51 show the nucleic acid and amino acid sequences of the heavy chain variable region VH of phage antibody clone L1H2, respectively. SEQ ID NO:63 and SEQ ID NO:47 show the nucleic acid and amino acid sequences of the light chain variable region VL of phage antibody clone L1H6, respectively. SEQ ID NO:67 and SEQ ID NO:52 show the nucleic acid and amino acid sequences of the heavy chain variable region VH of phage antibody clone L1H6, respectively. SEQ ID NO:64 and SEQ ID NO:48 show the nucleic acid and amino acid sequences of the light chain variable region VL of phage antibody clone L2H6, respectively. SEQ ID NO:67 and SEQ ID NO:52 show the nucleic acid and amino acid sequences of the heavy chain variable region VH of phage antibody clone L2H6, respectively.

[0336] Example 2. Expression and purification of intact anti-GPC3 antibody

[0337] Phage antibody clones 204A, L1H2, L1H6, and L2H6 were redesigned into complete IgG1,κ antibodies (i.e., the antigen-binding proteins isolated in this application). Specifically, primers were designed to amplify the VH phage antibody clone using PCR. The PCR product was then recombinantly cloned into the pCMV-IgG1NDL vector digested with AgeI and SalI. Primers were also designed to amplify the VL phage antibody clone using PCR. The PCR product was then recombinantly cloned into the pCMV-κ vector digested with AgeI and BsiWI. After successful sequencing, the heavy and light chain expression vectors were co-transfected into 293F cells for transient expression, and purified using a Protein A column to obtain complete IgG1,κ antibodies for 204A, L1H2, L1H6, and L2H6. SEQ ID NO:68 and SEQ ID NO:56 show the nucleic acid and amino acid sequences of the intact 204A antibody light chain; SEQ ID NO:71 and SEQ ID NO:59 show the nucleic acid and amino acid sequences of the intact 204A antibody heavy chain; SEQ ID NO:69 and SEQ ID NO:57 show the nucleic acid and amino acid sequences of the intact L1H2 antibody light chain; SEQ ID NO:72 and SEQ ID NO:60 show the nucleic acid and amino acid sequences of the intact L1H2 antibody heavy chain; SEQ ID NO:69 and SEQ ID NO:57 show the nucleic acid and amino acid sequences of the intact L1H6 antibody light chain; SEQ ID NO:73 and SEQ ID NO:61 show the nucleic acid and amino acid sequences of the intact L1H6 antibody heavy chain; SEQ ID NO:70 and SEQ ID NO:58 show the nucleic acid and amino acid sequences of the intact L2H6 antibody light chain; SEQ ID NO:73 and SEQ ID NO:61 show the nucleic acid and amino acid sequences of the intact L2H6 antibody heavy chain.

[0338] Example 3. Detection of binding affinity of intact anti-GPC3 antibody

[0339] The binding affinity of the intact antibodies 204A, L1H2, L1H6, and L2H6 obtained in Example 2 against recombinant human GPC3 protein was measured using an Octet RED384 instrument (Pall ForteBio). First, the recombinant human GPC3 protein (i.e., the antigen) was labeled with biotin (EZ-Link Sulfo-NHS-LC-Biotin, Pierce, 21327). The binding kinetics between the antigen and antibody were analyzed using a molecular interaction analyzer (fortebiooctetRED384 (PALL)) via biomembrane interference (BLI) technology (antigen and antibody dilutions were performed using 0.1% BSA and 0.02% Tween 20 in PBS buffer). The antigen was immobilized with a 50 nM biotin-conjugated SA sensor at 1500 rpm for 10 min; then, it was bound to a disproportionately diluted antibody solution (i.e., the intact antibodies 204A, L1H2, L1H6, and L2H6 obtained in Example 2) at 1500 rpm for 10 min. Finally, dissociation is performed for 10 minutes at 1500 rpm / min. Residual antibody will be regenerated via a glycine pulse. The results will be analyzed using OctetDataAnalysis 9.0 software (fortebio) to calculate the binding strength between the antigen and antibody, obtaining KB. D Values, Ka(1 / Ms) value, and Kd(1 / s) value. Anti-GPC3 intact antibodies 204A, L1H2, L1H6, and L2H6 all bind with high affinity to recombinant human GPC3 protein (as shown in Table 1).

[0340] Table 1. Binding affinity of intact anti-GPC3 antibody to human GPC3 protein

[0341]

[0342] In Table 1, Ka represents the binding rate constant; Kd represents the dissociation rate constant; and K represents the Kd. D : Affinity constant, equal to Kd / Ka.

[0343] Example 4. Epitope analysis of intact anti-GPC3 antibody

[0344] The specific epitope locations of L1H2, L1H6, and L2H6 antibodies were determined. Site-directed mutagenesis of the GPC3-His protein sequence was performed using PCR (Tiangen, KM101). The anti-GPC3 antibody showed no cross-reactivity with the mouse GPC3 antibody. Different sites in both wild-type human and mouse GPC3 proteins were mutated one-to-one with the corresponding mouse amino acids, such as... Figure 1As shown, there are a total of 11 sites. These 11 mutated GPC3-His proteins and GPC-His eukaryotic proteins were then expressed to obtain the corresponding proteins. The baselines of the above 12 proteins were adjusted to be consistent using Anti-6×His-HRP (Abcam, ab1187). Equal amounts of each of the 12 proteins were then coated at 1 μg / ml and incubated overnight at 4°C. The next day, after blocking with 5% skim milk powder (Sangon Biotech, A600669), primary antibodies L1H2, L1H6, L2H6, and GC33 antibodies (initial concentration 4 μg / ml, 4-fold serial dilution, 7 wells in total, the last well containing PBS) were added. The reaction was carried out for one hour. The secondary antibody was goat anti-human IgG HRP (Abcam, ab97225) at a dilution of 1:8000. GC33 represents the control antibody (see the patent document entitled "Anti-phosphatidylinositol proteoglycan 3 Antibody," publication number CN1842540 B). After processing at 37℃ for 45 min, and after thorough washing, TMB was added for color development. After processing at room temperature for 5-10 min, the reaction was terminated with sulfuric acid, and the OD value of each well was measured at 450 nm.

[0345] Statistical results are as follows Figure 2A-2B As shown in the results, when using wild-type human GPC3 protein, L1H2 antibodies bound slightly stronger than GC33 antibodies. When the phenylalanine of human GPC3 was mutated to serine (as shown in mutant 7), the binding of L1H2 and L2H6 to the GPC-His mutant protein was significantly weakened, while L1H6 and GC33 were less affected. When the proline of human GPC3 was mutated to glutamine (as shown in mutant 5), the binding of L2H6 to the GPC-His mutant protein was somewhat weakened, while the other three antibodies were less affected. This indicates that the epitope of L1H2 is located near phenylalanine, while the epitope of GC33 is located within the KDNEIST sequence at the C-terminus of GPC3.

[0346] Example 5. Detection of binding of intact anti-GPC3 antibody to cell surface GPC3

[0347] Fluorescence sorting (FACS) was performed using an iQue Screener flow cytometer (IntelliCyt) with PBS containing 0.1% BSA as buffer to detect the binding affinity of cell surface target antigen (GPC3) to antibodies (intact antibodies L1H2, L1H6, and L2H6 obtained in Example 2). The specific procedure is as follows:

[0348] 1. Prepare a buffer solution with a concentration of 1*10. 6Target cells (i.e., HepG2 liver cancer cells, Huh7 liver cancer cells, or L02 liver cells) per cell / ml were added to a 96-well conical plate (corning 3894), 30 μl per well.

[0349] 2. Prepare the detection antibody concentration to 3 μg / ml using buffer solution, and dilute the antibody 3-fold to form 8 concentration gradients;

[0350] 3. Add the prepared antibodies of different concentrations at a rate of 30 μl / well to the plated target cells and mix well;

[0351] 4. Incubate at 4°C for 1 hour;

[0352] 5. Add 150 μl of buffer to each well, centrifuge at 300g for 5 minutes, discard the supernatant and then loosen the cells by shaking.

[0353] 6. Repeat step 5;

[0354] 7. Prepare the fluorescent secondary antibody (ab98593) using buffer at a ratio of 1:200, add 30 μl to each well to the cells, mix well, and incubate at 4°C for 30 minutes;

[0355] 8. Add 150 μl of buffer to each well, centrifuge at 300g for 5 minutes, discard the supernatant and then loosen the cells by shaking.

[0356] 9. Repeat step 8;

[0357] 10. Add 35 μl of buffer solution to each well, mix well, and then detect using a flow cytometer;

[0358] 11. Use Graphpad software to analyze the data.

[0359] The results of the flow cytometry affinity binding assay are shown in Table 2, where GC33 represents the control antibody (see the patent document entitled Anti-phosphatidylinositol proteoglycan 3 Antibody, Publication No. CN1842540 B), and its expression and purification method is the same as in Example 2.

[0360] Table 2. Binding results of intact anti-GPC3 antibodies to GPC3 protein on cell surface

[0361]

[0362] As shown in Table 2, the flow cytometry binding capacity of L1H2, L2H6 and L1H6 intact antibodies on the surface of HepG2 liver cancer cells with high GPC3 expression and Huh7 liver cancer cells with moderate GPC3 expression was higher than that of GC33 antibody.

[0363] Example 6. Detection of ADCC activity of intact anti-GPC3 antibody

[0364] Anti-GPC3 intact antibodies (i.e., L1H2, L1H6, and L2H6 intact antibodies) target the ADCC activity of the GPC3-overexpressing hepatocellular carcinoma line HepG2 through... Luciferase detection system ( The Luciferase Assay System was used for testing.

[0365] The specific processing procedure for cell and antibody samples is as follows:

[0366] 1. HepG2 cells stably expressing luciferase (HepG2-luc) were cultured and amplified in DMEM (GIBICO) complete medium containing 10% FBS (GIBICO). After digestion of the cells with 0.25% trypsin (GIBICO), 1.95 x 10⁻⁶ cells were added. 6 Cells were resuspended in X-VIVO (Lonza) medium to 13 ml, forming 1.5 x 10⁻⁶ cells. 5 / ml of cell suspension is ready for use.

[0367] 2. Dilute 2 ml of apheresis blood three times with PBS to a final volume of 6 ml. Spread the diluted solution onto the top layer of 5 ml of ficoll (GE) separation buffer. Centrifuge at 500 g for 30 minutes, then collect the white membrane layer. Wash twice with 10 ml of PBS, centrifuging at 500 g for 5 minutes each time. Take 5 x 10 ml of the white membrane layer. 7 The obtained PBMC cells were resuspended in X-VIVO (Lonza) medium to 6.9 ml, forming 7.2 x 10⁻⁶ cells. 6 / ml of cell suspension is ready for use.

[0368] 3. Dilute the intact antibodies of L1H2, L1H6 and L2H6 to 30 μg / ml with X-VIVO (Lonza) medium, and then dilute them 10-fold successively to produce antibody solutions of 3 μg / ml, 0.3 μg / ml and 0.03 μg / ml for later use.

[0369] 4. Spread cells in a 96-well V-plate (Thermo) by adding 70 μl of HepG2-luc cell suspension, 70 μl of PBMC cell suspension, and 70 μl of each antibody solution to each well. Add two accessory wells to each group and incubate at 37°C in a CO2 incubator for 5 hours.

[0370] 5. 70 μl of HepG2-luc cell suspension, 70 μl of PBMC cell suspension, and 70 μl of X-VIVO medium in each well and 210 μl of X-VIVO medium in each well were used as non-killing control wells and background wells, respectively. Two sub-wells were added to each group and the cells were incubated in a CO2 incubator at 37°C for 5 hours.

[0371] Luciferase detection system ( The specific process of the Luciferase Assay system detection is as follows:

[0372] 1. After equilibrating the reagents to room temperature, use... Luciferase detection buffer ( Luciferase Assay Buffer) Resuspend Luciferase detection substrate ( LuciferaseAssay Substrate).

[0373] 2. Spread the resuspended test reagent at a rate of 100 μl / well into a 96-well flat-bottomed white plate (thermo).

[0374] 3. Remove the experimental plate from the 37℃ CO2 incubator and let it stand for 10 minutes to equilibrate to room temperature.

[0375] 4. Use a blowpipe to thoroughly resuspend the cells in each well to make them into a uniform cell suspension.

[0376] 5. Immediately transfer 100 μl of the cell suspension to the white plate containing the test reagent using a pipette and mix thoroughly by pipetting.

[0377] 6. Let stand in the dark for 10 minutes, then read the value using the luminescence detection program of the TECAN SPARK 10M ELISA reader.

[0378] 7. Analyze the readings:

[0379] Kill percentage = 100 × (no kill control wells - antibody test wells) / no kill control

[0380] Experimental results are as follows Figure 3 The results show that the complete antibodies L1H2, L1H6, and L2H6 can effectively mediate the killing of HepG2 liver cancer cell line with high GPC3 expression, i.e., they all have significant ADCC activity.

[0381] Example 7. Antitumor activity of L1H2 antibody against a mouse model of hepatocellular carcinoma transplantation

[0382] Huh7 cells were fed at a rate of 1×10 7Twenty BALB / c nude mice were subcutaneously injected with a 0.2 mL / vial solution on the right side of their tumor-bearing surface. On day 6 after tumor inoculation, the mice were randomly divided into two groups of eight (16 mice per tumor volume). Administration was via tail vein injection, administered four times daily on Days 0, 7, 14, and 28. Efficacy data were collected up to Day 41. The L1H2 antibody group received 10 mg / kg L1H2 via tail vein injection, while the control group received the same volume of PBS via tail vein injection. Mouse body weight and tumor volume were measured and recorded 2-3 times weekly during the administration and observation period. At the end of the experiment, the animals were euthanized, and changes in tumor volume were calculated.

[0383] In this experiment, the antitumor effect of L1H2 antibody on a mouse model transplanted with human hepatocellular carcinoma was evaluated by the change in tumor volume over time. Tumor volume was calculated using the following formula.

[0384] Tumor volume = (major axis) × (minor axis) × (minor axis) / 2

[0385] like Figure 4 As shown, compared with the control group, the L1H2 antibody group (L1H2 10 mg / kg IV) showed an inhibition rate of 59.67% against mouse tumor growth. Therefore, the antibody of this application has been shown to have anti-cancer activity in a mouse model of hepatocellular carcinoma transplantation.

[0386] The foregoing detailed description is provided by way of explanation and example and is not intended to limit the scope of the appended claims. Various variations of the embodiments listed herein will be apparent to those skilled in the art and are reserved within the scope of the appended claims and their equivalents.

Claims

1. An antibody or antigen-binding fragment thereof targeting GPC3, said antibody or antigen-binding fragment comprising an antibody light chain or a fragment thereof and an antibody heavy chain or a fragment thereof, said antibody light chain or fragment thereof comprising LCDR1, LCDR2, and LCDR3, said antibody heavy chain or fragment thereof comprising HCDR1, HCDR2, and HCDR3, wherein the amino acid sequences of said LCDR1, LCDR2, LCDR3, HCDR1, HCDR2, and HCDR3 are selected from the group consisting of: (1) HCDR1: SEQ ID NO: 12, HCDR2: SEQ ID NO: 13, HCDR3: SEQ ID NO: 14, LCDR1: SEQ ID NO: 7, LCDR2: SEQ ID NO: 8, LCDR3: SEQ ID NO: 9; or (2) HCDR1: SEQ ID NO: 12, HCDR2: SEQ ID NO: 13, HCDR3: SEQ ID NO: 14, LCDR1: SEQ ID NO: 7, LCDR2: SEQ ID NO: 15, LCDR3: SEQ ID NO:

9.

2. The antibody or antigen-binding fragment thereof according to claim 1, wherein the antibody light chain or fragment thereof comprises a light chain variable region VL, the antibody heavy chain or fragment thereof comprises a heavy chain variable region VH, and the amino acid sequences of VL and VH are selected from the group consisting of: (1) VL: SEQ ID NO:47, VH: SEQ ID NO:52; or (2) VL: SEQ ID NO:48, VH: SEQ ID NO:

52.

3. The antibody or its antigen-binding fragment according to claim 1, wherein the amino acid sequences of the antibody light chain and the antibody heavy chain are selected from the group consisting of: (1) Light chain: SEQ ID NO:57, heavy chain: SEQ ID NO:61; or (2) Light chain: SEQ ID NO:58, heavy chain: SEQ ID NO:

61.

4. One or more isolated nucleic acid molecules that encode an antibody or an antigen-binding fragment thereof as described in any one of claims 1-3.

5. A vector comprising the nucleic acid molecule according to claim 4.

6. A host cell comprising the nucleic acid molecule according to claim 4 or the vector according to claim 5.

7. A pharmaceutical composition comprising an antibody or an antigen-binding fragment thereof as described in any one of claims 1-3, a nucleic acid molecule as described in claim 4, a vector as described in claim 5, and / or a host cell as described in claim 6, and optionally a pharmaceutically acceptable adjuvant.

8. Use of the antibody or antigen-binding fragment thereof of any one of claims 1-3, the nucleic acid molecule of claim 4, the vector of claim 5, the host cell of claim 6, and / or the pharmaceutical composition of claim 7 in the preparation of a medicament for relieving and / or treating a tumor, wherein the tumor is liver cancer.

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